Method for treating cancer with a nanoformulation

ABSTRACT

A nanotherapeutic having platinum complexes encapsulated by a nanoformulation containing at least one spinel ferrite of formula CuFe 2 O 4 , NiFe 2 O 4 , CoFe 2 O 4 , and MnFe 2 O 4  deposited on mesoporous silica. A method of preparing the nanotherapeutic that involves mixing a metal(II) salt and a Fe(III) salt with the mesoporous silica nanoparticles to form a powdery mixture, calcining the powdery mixture to form the nanoformulation, and mixing the nanoformulation with the platinum complex. A method for treating cancer with the nanotherapeutic.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.16/394,543, now allowed, having a filing date of Apr. 25, 2019.

STATEMENT OF FUNDING ACKNOWLEDGEMENT

This project was funded by Deanship of Scientific Research (DSR), ImamAbdulrahman Bin Faisal University (IAU) under grant numbers2016-099-IRMC and 2016-072-DSR.

BACKGROUND OF THE INVENTION Technical Field

The present disclosure relates to a nanotherapeutic involving a spinelferrite impregnated mesoporous silica loaded with an anti-proliferativeplatinum complex. A method of preparing the nanotherapeutic, and amethod of treating cancer using the nanotherapeutic are also disclosed.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Cancer burden is expected to rise to 24 million in 2035 globally. Thecontinuing advancement of nanotechnology has increased chances to curechronic cancer, diabetes, and other metabolic disorders. Usingnanoparticles for targeted drug delivery, bioimaging, bioengineering,and stem cell applications may be a promising tactic for the treatmentof cancer [F. ud Din, W. Aman, I. Ullah, O. S. Qureshi, O. Mustapha, S.Shafique, A. Zeb, Effective use of nanocarriers as drug delivery systemsfor the treatment of selected tumors, Int J Nanomedicine 12 (2017)7291-7309]. An important feature of nanotherapeutics is the ability toaccommodate multiple components into a single nanostructure thatexhibits a variety of functions. However, challenges including lowbioavailability (about 5-10%), burst release, and low targeting accuracyneed to be addressed in order to develop an effective nano drug deliverysystem.

Multifunctional theranostic nanoparticles with imaging contrast agentshave attracted much attention in recent years. Magnetic nanosilica drugcarrier capable of responding to external magnetic field may assist inbioimaging (magnetic resonance imaging), magnetically targeted drugdelivery, enzyme lysozyme immobilization, hyperthermia, and tissuerepairs. Magnetic nanosilica has shown the potential to be loaded withcommercial cancer drugs.

In particular, superparamagnetic iron oxide nanoparticles (SPIONs) withintrinsic magnetic characteristics have been approved by FDA (U.S. foodand drug administration) for clinical uses [G. Jarockyte, E. Daugelaite,M. Stasys, U. Statkute, V. Poderys, T-C. Tseng, S-H. Hsu, V.Karabanovas, R. Rotomskis, Accumulation and Toxicity ofSuperparamagnetic Iron Oxide Nanoparticles in Cells and ExperimentalAnimals, Int J Mol Sci. 17 (2016) 1193]. Mesoporous silica (e.g. SBA-15(p6 mm)) containing magnetic Fe₃O₄ has been reported to be effective fordrug adsorption and delivery [Z. Vargas-Osorio, M. A. Gonzalez-Gomez, Y.Pineiro, C. Vazquez-Vazquez, C. Rodriguez-Abreu, M. A. Lopez-Quintela,J. Rivas, Novel synthetic routes of large-pore magnetic mesoporousnanocomposites (SBA-15/Fe₃O₄) as potential multifunctional theranosticnanodevices, J. Mater. Chem. B 5 (2017) 9395]. A biodegradablesilica/iron oxide nanocomposite with mesopores ranging from 20-60 nm wasreported for protein Ferritin delivery (cargo size >15 nm) [Omar H,Croissant J G, Alamoudi K, Alsaiari S, Alradwan I, Majrashi M A (2017)Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-LargeMesopores for High Protein Loading, Magnetothermal Release, andDelivery. J. Control. Release 259, 187-194]. However, SPIONs/silicabased nanostructures often have low saturation magnetization values. Forinstance, silica/iron oxide nanocomposite only showed a magnetizationvalue of 1.65 emu/g based on magnetometer (SQUID) analysis.

A recent study has demonstrated that loading SPIONs (10 wt %) ontosilica particles with various structures could induce different levelsof magnetization. For example, SPIONs loaded micron-sized sphericalsilica exhibited a highest magnetization value of 1.44 emu/g, whileSPIONs loaded silicalite particles showed a lowest value of 0.08 emu/g[B. R. Jermy, V. Ravinayagam, S. Akhtar, W. A. Alamoudi, N. A. Alhamed,A. Baykal. Magnetic Mesocellular Foam Functionalized by Curcumin forPotential Multifunctional Therapeutics, J Supercond Nov Magn (2018).https://doi.org/10.1007/s10948-018-4921-3]. Such variation could beattributed to the presence of large surface area of silica that tends toconvert SPIONs into nano-sized particles (3-21 nm) rather thancrystalline oxides. In general, a large magnetization value is requiredfor a nanoformulation to perform multiple tasks effectively.Magnetization is expected to increase by loading a larger amount ofSPIONs onto the nanoformulation. However, a large loading of SPIONscould lead to formation of mixtures of iron oxide species (α-Fe₂O₄,Fe₃O₄ and γ-Fe₂O₄).

Spinel ferrites may be formed by modifying the surface of iron oxidewith transition heteroatoms (M). Magnetic nanoferrites are inexpensiveand can be easily prepared without multistep protocols. Ferromagnetismmay occur when anti parallel spins of Fe³⁺ are present at tetrahedralsites, and transition heteroatoms (M²⁺) occupy octahedral sites.Recently, a low-cost preparation of nano copper ferrite using citratesol gel technique was reported [Md. Amir, H. Gungunes, Y. Silmani, N.Tashkandi, H. S. El Sayed, F. Aldakheel, M. Sertkol, H. Sozeri, A.Manikandan, I. Ercan, A. Baykal, Journal of Superconductivity and NovelMagnetism, https://doi.org/10.1007/s10948-018-4733-5]. In addition,several metal-based ferrite systems have been reported lately forbiomedical applications such as hyperthermia, magnetic resonanceimaging, and drug delivery [M. R. Phadatare, V. M. Khot, A. B. Salunkhe,N. D. Thorat, S. H. Pawar, Studies on polyethylene glycol coating onNiFe₂O₄ nanoparticles for biomedical applications, Journal of Magnetismand Magnetic Materials, 324 (2012) 770-772; and I. Sharifi, H.Shokrollahi, S. Amiri, Ferrite-based magnetic nanofluids used inhyperthermia applications, Journal of Magnetism and Magnetic Materials324 (2012) 903-915, each incorporated herein by reference in theirentirety].

However, spinel ferrite nanoparticles tend to form aggregations due tostrong magnetic dipole-dipole interactions. Silica- and carbon-basedsupports were used to reduce the aggregation. Notably, different typesof nanocomposite having Co-, Ni-, Mn- and Fe-based ferrites depositedover mesoporous carbon capsules as supports were reported by Fuertes etal. [A. B. Fuertes, T. Valdes-Solis, M. Sevilla, Fabrication ofMonodisperse Mesoporous Carbon Capsules Decorated with FerriteNanoparticles, J. Phys. Chem. C 2008, 112, 3648-3654, incorporatedherein by reference in its entirety]. Carbon capsule supports with ashell thickness of about 50 nm demonstrated a high loading capacity offerrites at about 30-50 wt %. Ferrites with a particle size between 9-17nm were observed at external carbon layers.

The magnetic properties of ZnFe₂O₄/MCM-41 and NiFe₂O₄/MCM-41 synthesizedthrough wet impregnation technique have been studied and compared withbare ferrites. The findings showed that ferrites enclosed inside thenon-magnetic hexagonal pores of MCM-41 exhibited smaller dipolarinteractions and reduced magnetization value due to surface anisotropyeffect [M. Virumbrales, R. Saez-Puche, M. José Torralvo, V.Blanco-Gutierrez, Mesoporous Silica Matrix as a Tool for MinimizingDipolar Interactions in NiFe₂O₄ and ZnFe₂O₄ Nanoparticles, Nanomaterials2017, 7, 151, incorporated herein by reference in its entirety].

Bullita et al. [S. Bullita, A. Casu, M. F. Casula, G. Concas F. Congiu,A. Corrias A. Falqui, D. Loche and C. Marras, ZnFe₂O₄ nanoparticlesdispersed in a highly porous silica aerogel matrix: a magnetic study,Phys. Chem. Chem. Phys., 2014, 16, 4843, incorporated herein byreference in its entirety] reported the superparamagnetic effect of azinc ferrite system over aerogel. The effect of calcination of zincferrite/aerogel nanocomposite at different temperatures between 450-900°C. in static air (450° C. for 1 h, 750° C. for 1 h and 6 h, 900° C. for1 h) was studied. It was found that the thermal treatment temperaturemight directly impact the particle size of ZnFe₂O₄, as well as theinversion degree of normal bulk spinel ferrite. MesoporousCu_(1-x)Zn_(x)Fe₂O₄ system has been reported using nanocasting technique[N. Najmoddin, A. Beitollahi, H. Kavas, S. M. Mohseni, H. Rezaie, J.Åkerman, M. S. Toprak, XRD cation distribution and magnetic propertiesof mesoporous Zn-substituted CuFe₂O₄, Ceramics International 40 (2014)3619-3625, incorporated herein by reference in its entirety]. Based onBertaut analysis, doping of Zn tends to form mixed inverse spinels whereZn occupies the A site, while Cu prefers to occupy the B site. SQUID-VSManalysis observed superparamagnetic behavior of the mixed metal oxidespinel composite. Temperature dependence study using field cooling andzero field cooling analysis (ZFC/FC) showed spin glass like surfacelayers in the mixed metal oxide spinel composite. CuFe₂O₄ and activatedcarbon composite prepared using co-precipitation technique was reportedby [G. Zhang, J. Qu, H. Liu, A. T. Cooper, R. Wu, CuFe₂O₄/activatedcarbon composite: A novel magnetic adsorbent for the removal of acidorange II and catalytic regeneration, Chemosphere 68 (2007) 1058-1066,incorporated herein by reference in its entirety]. The study showed thatthe presence of spinel over carbon support was useful for adsorption andhad minimal impact on the surface area occupation and pore sizedistribution. Hammiche-Bellal et al. [Y. Hammiche-Bellal, N.Zouaoui-Mahzoul, I. Lounas, A. Benadda, R. Benrabaa, A. Auroux, L.Meddour-Boukhobza, A. Djadoun, Cobalt and cobalt-iron spinel oxides asbulk and silica supported catalysts in the ethanol combustion reaction,Journal of Molecular Catalysis A: Chemical 426 (2017) 97-106,incorporated herein by reference in its entirety] studied the effect ofcobalt and cobalt ferrite on silica and bulk support throughimpregnation and co-precipitation techniques. Mixed metal oxides ofspinels showed highly active sites and a uniform dispersion was found onthe silica surface rather than bulk surface.

Briefly, the synthesis and characterization of Cu, Ni, Co and Mn spinelferrites have been carried out. However, nanoformulations having Cu, Ni,Co and Mn spinel ferrites deposited on monodispersed hydrophilic silica(HYPS) for drug adsorption and delivery has not been explored.Furthermore, studies regarding adsorption, releasing, and toxicity ofcancer drug (e.g. cisplatin) loaded spinel ferrite/monodispersedhydrophilic has not been conducted.

In view of the forgoing, one objective of the present disclosure is toprovide a nanotherapeutic involving a platinum complex loadednanoformulation that contains a spinel ferrite (e.g. CuFe₂O₄, NiFe₂O₄,CoFe₂O₄, MnFe₂O₄) impregnated mesoporous silica. A further objective ofthe present disclosure is to provide a method of making thenanotherapeutic and a method of treating cancer by administrating thenanotherapeutic. The nanotherapeutic demonstrates magnetic activity andexhibit efficient loading and releasing capability of platinum complexeswith anticancer efficacy.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present disclosure relates to ananotherapeutic involving i) a nanoformulation that contains mesoporoussilica nanoparticles and a spinel ferrite of formula (I)MFe₂O₄  (I)and ii) a platinum complex encapsulated within pores of thenanoformulation, wherein M is at least one transition metal elementselected from the group consisting of Cu, Ni, Co, and Mn, the spinelferrite is impregnated on the mesoporous silica nanoparticles, and thespinel ferrite is present in an amount of 15-50 wt % relative to a totalweight of the nanoformulation.

In one embodiment, the spinel ferrite of formula (I) is CuFe₂O₄,NiFe₂O₄, or both.

In one embodiment, the pores of the nanoformulation have a pore diameterin a range of 10-25 nm.

In one embodiment, the nanoformulation has a pore volume in a range of0.05-0.3 cm³/g.

In one embodiment, the nanoformulation has a BET surface area in a rangeof 15-70 m²/g,

In one embodiment, the nanoformulation has a saturation magnetizationvalue in a range of 1-18 emu/g.

In one embodiment, the platinum complex is present at a concentration of0.01-10 mmol/g relative to a total weight of the nanoformulation.

In one embodiment, the platinum complex is at least one selected fromthe group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin,triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.

In one embodiment, the platinum complex is cisplatin.

In one embodiment, the nanotherapeutic further comprises one or morepharmaceutically acceptable carriers and/or excipients selected from thegroup consisting of a buffer, an inorganic salt, a fatty acid, avegetable oil, a synthetic fatty ester, a surfactant, and a polymer.

According to a second aspect, the present disclosure relates to a methodof preparing the nanotherapeutic of the first aspect. The methodinvolves the steps of i) mixing an M(II) salt and a Fe(III) salt withthe mesoporous silica nanoparticles to form a powdery mixture, ii)calcining the powdery mixture to form a nanoformulation, and iii) mixingthe nanoformulation and a platinum complex in an aqueous solution,thereby forming the nanotherapeutic.

In one embodiment, the calcining is performed at a temperature of600-1,000° C.

In one embodiment, the aqueous solution is saline.

In one embodiment, the platinum complex is present in the aqueoussolution at a concentration of 0.1-30 g/L, and the nanoformulation ispresent in the aqueous solution at a concentration of 2-600 g/L, eachrelative to a total volume of the aqueous solution.

According to a third aspect, the present disclosure relates to a methodfor treating a proliferative disorder. The method involves administeringthe nanotherapeutic of the first aspect to a subject in need of therapy.

In one embodiment, 0.5-800 mg/kg of the nanotherapeutic is administeredper body weight of the subject.

In one embodiment, the platinum complex is cisplatin.

In one embodiment, the proliferative disorder is a cancer selected fromthe group consisting of breast cancer, ovarian cancer, cervical cancer,testicular cancer, colon cancer, bladder cancer, and lung cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the subject is a mammal.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows X-ray diffraction (XRD) patterns of nanoformulationcontaining copper ferrite (CuFe₂O₄).

FIG. 2 is an overlay of XRD patterns of nanoformulations containingcopper ferrite with various copper contents.

FIG. 3 is an overlay of XRD patterns of nanoformulations containingcopper ferrite calcined at a temperature of 700° C., 750° C., 800° C.,850° C., and 900° C., respectively.

FIG. 4A is an overlay of N₂ sorption isotherms of mesoporous silicananoparticles (HYPS) and nanoformulation containing copper ferrite (30wt % CuFe₂O₄/HYPS), respectively.

FIG. 4B is a graph showing pore size distributions of mesoporous silicananoparticles (HYPS) and nanoformulation containing copper ferrite (30wt % CuFe₂O₄/HYPS), respectively.

FIG. 5A is a scanning electron microscope (SEM) image of mesoporoussilica nanoparticles.

FIG. 5B is a SEM image of nanoformulation containing copper ferrite.

FIG. 5C is an energy-dispersive spectroscopy (EDS) layered image ofnanoformulation containing copper ferrite.

FIG. 5D is an EDS spectrum of nanoformulation containing copper ferrite.

FIG. 5E is an elemental mapping of iron of nanoformulation containingcopper ferrite.

FIG. 5F is an elemental mapping of copper of nanoformulation containingcopper ferrite.

FIG. 6 is an overlay of FT-IR spectra of mesoporous silica nanoparticles(HYPS) and nanoformulation containing copper ferrite (30 wt %CuFe₂O₄/HYPS), respectively.

FIG. 7 is an overlay of magnetization curves of nanoformulationscontaining CuFe₂O₄, NiFe₂O₄, MnFe₂O₄, and CoFe₂O₄, respectively obtainedby vibrating sample magnetomer (VSM).

FIG. 8 is a bar graph showing the cisplatin adsorption/cumulativerelease (%) over 24 hours at pH 5 as well as cisplatin loading capacity(mmol cisplatin/1 g of HYPS) of nanotherapeutics containing CuFe₂O₄,NiFe₂O₄, MnFe₂O₄, and CoFe₂O₄, respectively.

FIG. 9 shows cisplatin release profiles of nanotherapeutics containingCuFe₂O₄, NiFe₂O₄, MnFe₂O₄, and CoFe₂O₄, respectively, over 72 hoursunder simulated tumor acidic conditions (i.e. pH 5, 37° C.).

FIG. 10 is an overlay of UV-vis diffuse reflectance spectroscopy (DRS)spectra of nanoformulations containing CuFe₂O₄ (a), nanotherapeuticcontaining CuFe₂O₄ (b), nanoformulations containing MnFe₂O₄ (c), andnanotherapeutic containing MnFe₂O₄ (d), respectively.

FIG. 11 shows MCF-7 cell viability MTT assay results of CuFe₂O₄ (groupA), mesoporous silica nanoparticles (group B), nanoformulationcontaining CuFe₂O₄ (group C), cisplatin (group D), nanotherapeuticcontaining CuFe₂O₄ (group E), and nanotherapeutic containing CuFe₂O₄ andchitosan (group F), respectively.

FIG. 12A is a graph showing MCF-7 cell viability percentage underdifferent concentrations of CuFe₂O₄ (group A).

FIG. 12B is a graph showing MCF-7 cell viability percentage underdifferent concentrations of mesoporous silica nanoparticles (group B).

FIG. 12C is a graph showing MCF-7 cell viability percentage underdifferent concentrations of nanoformulation containing CuFe₂O₄ (groupC).

FIG. 12D is a graph showing MCF-7 cell viability percentage underdifferent concentrations of cisplatin (group D).

FIG. 12E is a graph showing MCF-7 cell viability percentage underdifferent concentrations of nanotherapeutic containing CuFe₂O₄ (groupE).

FIG. 12F is a graph showing MCF-7 cell viability percentage underdifferent concentrations of nanotherapeutic containing CuFe₂O₄ andchitosan (group F).

FIG. 12G is a table summarizing EC₅₀ values of CuFe₂O₄ (group A),mesoporous silica nanoparticles (group B), nanoformulation containingCuFe₂O₄ (group C), cisplatin (group D), nanotherapeutic containingCuFe₂O₄ (group E), and nanotherapeutic containing CuFe₂O₄ and chitosan(group F), respectively, against MCF-7 cell lines.

FIG. 13 is a schematic representation of drug delivery mechanism ofnanotherapeutic containing CuFe₂O₄.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

The present disclosure will be better understood with reference to thefollowing definitions. As used herein, the words “a” and “an” and thelike carry the meaning of “one or more”. Within the description of thisdisclosure, where a numerical limit or range is stated, the endpointsare included unless stated otherwise. Also, all values and subrangeswithin a numerical limit or range are specifically included as ifexplicitly written out.

As used herein, the terms “complex”, “compound”, and “product” are usedinterchangeably, and are intended to refer to a chemical entity, whetherin the solid, liquid or gaseous phase, and whether in a crude mixture orpurified and isolated.

The present disclosure includes all hydration states of a given salt orformula, unless otherwise noted. For example, copper(II) nitrateincludes anhydrous Cu(NO₃)₂, monohydrate Cu(NO₃)₂.H₂O,hemi(pentahydrate) Cu(NO₃)₂.2.5H₂O, trihydrate Cu(NO₃)₂.3H₂O, and anyother hydrated forms or mixtures. Iron(III) nitrate includes anhydrousFe(NO₃)₃, and hydrated forms such as iron(III) nitrate nonahydrateFe(NO₃)₃.9H₂O.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample, and without limitation, isotopes of hydrogen include deuteriumand tritium, isotopes of carbon include ¹²C, ¹³C, and ¹⁴C, isotopes ofoxygen include ¹⁶O, ¹⁷O, and ¹⁸O, isotopes of copper include ⁶³Cu and⁶⁵Cu, and isotopes of iron include ⁵⁴Fe, ⁵⁶Fe, ⁵⁷Fe, and ⁵⁸Fe.Isotopically labeled compounds of the disclosure can generally beprepared by conventional techniques known to those skilled in the art orby processes and methods analogous to those described herein, using anappropriate isotopically labeled reagent in place of the non-labeledreagent otherwise employed.

According to a first aspect, the present disclosure relates to ananotherapeutic involving i) a nanoformulation that contains mesoporoussilica nanoparticles and a spinel ferrite of formula (I)MFe₂O₄  (I)and ii) a platinum complex encapsulated within pores of thenanoformulation.

A “mesoporous support” refers to a porous support material with largestpore diameters ranging from about 2-50 nm, preferably 3-45 nm,preferably 4-40 nm, preferably 5-25 nm. As used herein, “mesoporoussilica” refers to a mesoporous support comprising silica (SiO₂).Non-limiting examples of mesoporous silica include MCM-48, MCM-41,MCM-18, SBA-15, and SBA-16.

A particle is defined as a small object that behaves as a whole unitwith respect to its transport and properties. An average diameter (e.g.,average particle size) of the particle, as used herein, and unlessotherwise specifically noted, refers to the average linear distancemeasured from one point on the particle through the center of theparticle to a point directly across from it. For a circle, an oval, anellipse, and a multilobe, the term “diameter” refers to the greatestpossible distance measured from one point on the shape through thecenter of the shape to a point directly across from it. For polygonalshapes, the term “diameter”, as used herein, and unless otherwisespecified, refers to the greatest possible distance measured from avertex of a polygon through the center of the face to the vertex on theopposite side. The mesoporous silica support used herein may be in theform of particles (i.e. mesoporous silica particles). In one embodiment,the mesoporous silica particles have an average particle size of 0.01-3μm, 0.05-2 μm, 0.1-1 μm, or 0.2-0.5 μm.

Nanoparticles are particles between 1 and 100 nm in size. In preferredembodiments, the mesoporous silica support of the present disclosure ispresent in the form of nanoparticles. In a preferred embodiment, themesoporous silica nanoparticles have an average particle size of 10-100nm, 25-95 nm, 30-90 nm, 40-85 nm, 50-85 nm, 60-80 nm, or 70-75 nm. Themesoporous silica nanoparticles may preferably be spherical orsubstantially spherical (e.g., oval or oblong shape). In otherembodiments, the mesoporous silica nanoparticles can be of any shapethat provides desired permeability and/or stability of thenanoformulation, and/or release rates of encapsulated compounds (e.g.,the platinum complex). For example, the mesoporous silica nanoparticlesmay be in a form of at least one shape such as a sphere, a rod, apentagon, a hexagon, a prism, a disc, and a platelet.

Dispersity is a measure of the heterogeneity of sizes of particles in amixture. In probability theory and statistics, the coefficient ofvariation (CV) also known as relative standard deviation (RSD) is astandardized measure of dispersion of a probability distribution. It isexpressed as a percentage and is defined as the ratio of the standarddeviation (σ) of to the mean (μ, or its absolute value |μ|). The CV orRSD is widely used to express precision and repeatability. It shows theextent of variability in relation to the mean of a population.Preferably, the mesoporous silica nanoparticles have a narrow sizedispersion, i.e. monodispersity. As used herein, “monodisperse”,“monodispersed” and/or “monodispersity” refers to nanocapsules having aCV or RSD of less than 25%, preferably less than 20%.

The mesoporous silica nanoparticles may be monodisperse with acoefficient of variation or relative standard deviation (ratio of theparticle size standard deviation to the particle size mean) of less than15%, less than 12%, less than 10%, less than 9%, less than 8%, less than7%, less than 6%, less than 5%, or preferably less than 2%. In oneembodiment, the mesoporous silica nanoparticles are monodisperse andhave a particle diameter distribution in a range of 75% of the averageparticle diameter to 125% of the average particle diameter, 80-120%,85-115%, 86-114%, 87-113%, 88-112%, 89-111%, 90-110%, or preferably95-105% of the average particle diameter. Alternatively, the mesoporoussilica nanoparticles are polydisperse with a coefficient of variation orrelative standard deviation (ratio of the particle size standarddeviation to the particle size mean) of more than 15%, 20%, or 30%. Thepolydisperse nanoparticles may have a particle diameter distribution ina range of 25% of the average particle diameter to 175% of the averageparticle diameter, 30-160%, or 50-150% of the average particle diameter.

The silica nanoparticles may be agglomerated or non-agglomerated (i.e.,the nanoparticle are well separated from one another and do not formclusters). In some embodiments, the silica nanoparticles may cluster andform agglomerates having an average diameter in a range of 2-50 μm, 4-25μm, or 5-10 μm.

The Brunauer-Emmet-Teller (BET) theory (S. Brunauer, P. H. Emmett, E.Teller, J. Am. Chem. Soc. 1938, 60, 309-319, incorporated herein byreference) aims to explain the physical adsorption of gas molecules on asolid surface and serves as the basis for an important analysistechnique for the measurement of a specific surface area of a material.Specific surface area is a property of solids which is the total surfacearea of a material per unit of mass, solid or bulk volume, or crosssectional area. The surface area and pore size distribution may becharacterized using a method developed by Barrett, Joyner and Halenda(BJH) (E. P. Barrett, L. G. Joyner, P. P. Halenda, J. Am. Chem. Soc.1951, 73, 373-380, incorporated herein by reference) through gasadsorption analysis. In preferred embodiments, pore volume and BETsurface area are measured by gas adsorption analysis, preferably N₂adsorption analysis.

In one or more embodiments, the mesoporous silica nanoparticles have apore volume of 0.2-0.5 cm³/g, 0.3-0.45 cm³/g, 0.32-0.4 cm³/g, or about0.35 cm³/g. In one related embodiment, the mesoporous silicananoparticles have a pore diameter of 2-10 nm, 4-9 nm, 5-8.5 nm, orabout 8.3 nm. In another related embodiment, the mesoporous silicananoparticles have a BET surface area of 80-400 m²/g, 100-300 m²/g,120-250 m²/g, 150-200 m²/g, or about 170 m²/g.

In certain embodiments, the mesoporous silica support used herein haspore channels that are regularly arranged. For example, the mesoporoussilica support may be in the form of a honeycomb-like structure havingpore channels parallel or substantially parallel to each other within atwo-dimensional hexagon (e.g. SBA-15, MCM-41). Alternatively, othermesoporous silica structures such as SBA-11 having a cubic structure,SBA-12 having a three-dimensional hexagonal structure, and SBA-16 havinga cubic in cage-like structure may be used as the mesoporous silicasupport. The mesoporous silica nanoparticles may be available fromcommercial vendors including, without limitation, Superior Silica, SigmaAldrich, and Alfa Aesar.

As defined herein, a spinel is a metal oxide compound with a generalformula A²⁺B₂ ³⁺O₄ ²⁻, where “A” and “B” are metal ions. In oneembodiment, “A” may be Zn, Cu, Co, Mn, Ni, Mg, Be, and/or Ti, and “B”may be Al, Fe, Cr, and/or V. Preferably, spinel compounds are in theform of crystals, with the oxide anions arranged in a cubic close-packedlattice, and with the metal ions occupying octahedral and/or tetrahedralsites within the lattice. Preferably, the A²⁺ metal ions occupy thetetrahedral sites, and the B³⁺ metal ions occupy the octahedral sites,though there may be instances where the metal ions are switched. The A²⁺and B³⁺ metal ions may occupy sites in the lattice at regular spacingsor may be distributed randomly.

A spinel ferrite is defined herein as an iron-containing spinel compoundwith a formula (I)MFe₂O₄  (I)where “M” is a metal ion. In one embodiment, “M” may be Zn, Cu, Co, Mn,Ni, Mg, Be, and/or Ti. In a preferred embodiment, M is at least onetransition metal element selected from the group consisting of Cu, Ni,Co, and Mn. Most preferably, the spinel ferrite of formula (I) isCuFe₂O₄, NiFe₂O₄, or both.

In certain embodiments, the spinel ferrite of the present disclosure isa mixed spinel ferrite with a formula M_(x) ²⁺N_((1-x)) ²⁺Fe₂O₄, where“M” is the same as previously described, and “N” may be a metal ion(e.g. Zn, Cu, Co, Mn, Ni, Mg, Be, Ti) that is different from ‘M”, and xis greater than 0 and smaller than 1. Atomic ratios of the mixed spinelferrites may be determined by elemental analysis techniques such asenergy-dispersive X-ray spectroscopy (EDX), X-ray photoelectronspectroscopy (XPS), inductively coupled plasma mass spectrometry(ICP-MS), and neutron activation analysis.

In preferred embodiments, the spinel ferrite disclosed herein is copperferrite CuFe₂O₄, nickel ferrite (NiFe₂O₄), a mixed spinel ferrite havingoxygen atoms and metal atoms including copper and nickel (i.e.Cu_(x)Ni_((1-x))Fe₂O₄, where 0<x<1), or a mixture thereof. In at leastone embodiment, the spinel ferrite is devoid of other metal atoms suchas cobalt (Co) and manganese (Mn).

The nanoformulation of the present disclosure comprises theaforementioned spinel ferrite impregnated on the mesoporous silicananoparticles. As used herein, “impregnated” or “disposed on” describesbeing partially filled throughout, saturated, permeated, and/or infused.The spinel ferrite may be affixed on one or more surfaces of themesoporous silica nanoparticles. For example, the spinel ferrite may beaffixed on an outer surface of the mesoporous silica nanoparticles, orwithin pore spaces of the mesoporous silica nanoparticles. Preferably,the spinel ferrite is affixed to external pores of the mesoporous silicananoparticles. The spinel ferrite may be affixed to the mesoporoussilica nanoparticles in any reasonable manner, such as physisorption,chemisorption, or combinations thereof. In one embodiment, up to 10% ofthe surface area (i.e. outer surface and pore spaces) of the mesoporoussilica nanoparticles is covered by the spinel ferrite. Preferably up to15%, preferably up to 20%, preferably up to 25%, preferably up to 30%,preferably up to 35%, preferably up to 40%, preferably up to 45%,preferably up to 50%, preferably up to 75% of the surface area of themesoporous silica nanoparticles is covered by the spinel ferrite. Inpreferred embodiments, at least 25% of the surface area of themesoporous silica nanoparticles is not covered by the spinel ferrite,and thus the nanoparticles are available for encapsulating othercompounds (e.g. platinum complexes). Preferably at least 30%, preferablyat least 40%, preferably at least 50%, preferably at least 55%,preferably at least 60%, preferably at least 65%, preferably at least70%, preferably at least 75%, preferably at least 80%, preferably atleast 85% of the surface area of the mesoporous silica nanoparticles isnot covered by the spinel ferrite, and thus the nanoparticles areavailable for encapsulating other compounds (e.g. platinum complexes).

In one or more embodiments, the spinel ferrite is present in an amountof 15-50 wt %, preferably 20-40 wt %, preferably 25-35 wt %, or about 30wt % relative to a total weight of the nanoformulation. However, incertain embodiments, the spinel ferrite is present in an amount lessthan 15 wt % or greater than 50 wt % relative to a total weight of thenanoformulation.

In one or more embodiments, the pores of the nanoformulation of thepresent disclosure have a pore diameter in a range of 10-25 nm,preferably 12-20 nm, more preferably 15-18 nm. In one relatedembodiment, the nanoformulation has a pore volume in a range of 0.05-0.3cm³/g, preferably 0.08-0.2 cm³/g, more preferably 0.1-0.18 cm³/g. Inanother related embodiment, the nanoformulation has a BET surface areain a range of 15-70 m²/g, preferably 20-60 m²/g, more preferably 30-50m²/g.

The nanoformulation disclosed herein may be paramagnetic, ferromagnetic,or superparamagnetic. In one embodiment, the nanoformulation comprisingcopper ferrite (CuFe₂O₄) may be paramagnetic that is weakly attracted byan externally applied magnetic field and form induced magnetic fields inthe direction of the applied magnetic field. In another embodiment, thenanoformulation comprising cobalt ferrite (CoFe₂O₄) may be ferromagneticcontaining populations of atoms with aligned magnetic moments. Inanother embodiment, the nanoformulation comprising nickel ferrite(NiFe₂O₄) and/or manganese ferrite (MnFe₂O₄) may show superparamagnetismwhich is a form of magnetism appearing in ferromagnetic or ferrimagneticnanoparticles. In sufficiently small particles, such as thenanoformulation described herein, magnetization can randomly flipdirection under the influence of temperature. In the absence of anexternal magnetic field, the magnetization appears to be zero and thenanoformulation is in the superparamagnetic state. In this state, anexternal magnetic field is able to magnetize the nanoformulation.Superparamagnetic materials have a magnetic susceptibility larger thanthat of paramagnets.

The nanoformulation disclosed herein in any of its embodiments may havea saturation magnetization value in a range of 1-20 emu/g, 5-18 emu/g,7-15 emu/g, or 9-12 emu/g (see FIG. 7 ). The magnetic susceptibilitiesmay be measured with a laboratory magnetometer such as a vibratingsample magnetometer (VSM), a superconducting quantum interferencedevice, inductive pickup coils, a pulsed field extraction magnetometer,a torque magnetometer, a faraday force magnetometer, and an opticalmagnetometer.

In one or more embodiments, the nanotherapeutic of the presentdisclosure comprises a platinum complex encapsulated within pores of theaforementioned nanoformulation. The nanoformulation may be loaded withany platinum complex effective for the treatment of cancer. Preferably,the platinum complex is a platinum(II) complex. In one or moreembodiments, the platinum complex is at least one selected from thegroup consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin,triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.In a preferred embodiment, the platinum complex is at least one ofcisplatin, carboplatin, oxaliplatin, and nedaplatin. Most preferably,the platinum complex is cisplatin.

Cisplatin is an anticancer drug that binds to the DNA blocking celldivision [S. Dasari, P. B. Tchounwou. Cisplatin in cancer therapy:molecular mechanisms of action. Eur J Pharmacol. 740 (2014) 364-378].Similar to many other anticancer drugs, cisplatin has off targettoxicities that mainly occur in the kidneys, liver, heart, nerves, andears [L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O.Kepp, M. Castedo, G. G. Kroemer. Molecular mechanisms of cisplatinresistance. Oncogene 31 (2012) 1869-1883; and S. Dasari, P. B.Tchounwou. Cisplatin in cancer therapy: molecular mechanisms of action.Eur J Pharmacol. 740 (2014) 364-378, each incorporated herein byreference in their entirety]. In addition, most patients developchemoresistance to cisplatin [L. Galluzzi, L. Senovilla, I. Vitale, J.Michels, I. Martins, O. Kepp, M. Castedo, G. G. Kroemer. Molecularmechanisms of cisplatin resistance. Oncogene 31 (2012) 1869-1883].Cisplatin-loaded/CuFe₂O₄-coated silica nanoparticles may help overcomethese limitations and ensure specific tumor targeting. The encapsulationof cisplatin may prevent off target delivery.

The platinum complex may be encapsulated by the nanoformulation andoptionally fill at least some of the pores of the nanoformulation. Theplatinum complex may be adsorbed onto the surface and/or within thepores of the nanoformulation via physisorption and/or chemisorptioninteractions such as hydrogen bonds (e.g. Cl . . . H interaction, forplatinum complex containing chlorine groups), electrostatic forces, andvan der Waals forces.

The platinum complex may be present at a concentration of 0.01-10 mmol/grelative to a total weight of the nanoformulation. Preferably theplatinum complex is present at a concentration of 0.05-5 mmol/g,preferably 0.1-2.5 mmol/g, preferably 0.12-2 mmol/g, preferably0.13-0.18 mmol/g, preferably 0.14-0.17 mmol/g, preferably 0.15-0.16mmol/g relative to a total weight of the nanoformulation. However, incertain embodiments, the platinum complex is present at a concentrationless than 0.01 mmol/g or greater than 10 mmol/g relative to a totalweight of the nanoformulation.

The present disclosure further relates to a method of preparing thenanotherapeutic. The method involves the steps of i) mixing an M(II)salt and a Fe(III) salt with the mesoporous silica nanoparticles to forma powdery mixture, ii) calcining the powdery mixture to form ananoformulation, and iii) mixing the nanoformulation and a platinumcomplex in an aqueous solution, thereby forming the nanotherapeutic.

Non-limiting examples of the Fe(III) salt include iron(III) nitrate,iron(III) chloride, iron(III) sulfate, iron(III) bromide, iron(III)fluoride, iron(III) phosphate, and mixtures thereof. The iron(III) saltused herein may be in any hydration state, for instance, iron(III)nitrate includes, without limitation, Fe(NO₃)₃, Fe(NO₃)₃.6H₂O, andFe(NO₃)₃.9H₂O. In certain embodiments, an iron salt having a differentoxidation state, such as +2, may be used in addition to or in lieu ofthe iron(III) salt. In a preferred embodiment, the Fe(III) salt isiron(III) nitrate.

Depending on the chemical identity of “M” of the spinel ferrite intendedto be present in the nanotherapeutic, the M(II) salt may be Zn(II),Cu(II), Co(II), Mn(II), Ni(II), Mg(II), Be(II), and/or Ti(II).Preferably, the M(II) salt is at least one selected from the groupconsisting of Cu(II), Co(II), Mn(II), and Ni(II). Most preferably, theM(II) salt is Cu(II), Ni(II), or both.

Non-limiting examples of the Cu(II) salt include copper(II) nitrate,copper(II) chloride, copper(II) sulfate, copper(II) bromide, copper(II)iodide, and mixtures thereof. The copper(II) salt used herein may be inany hydration state, for instance, copper(II) nitrate includes, withoutlimitation, Cu(NO₃)₂, Cu(NO₃)₂.H₂O, Cu(NO₃)₂.2.5H₂O, Cu(NO₃)₂.3H₂O, andCu(NO₃)₂.6H₂O. In certain embodiments, a copper salt having a differentoxidation state, such as +1, may be used in addition to or in lieu ofthe copper(II) salt. In a preferred embodiment, the Cu(II) salt iscopper(II) nitrate.

Non-limiting examples of the Ni(II) salt include nickel(II) nitrate,nickel(II) chloride, nickel(II) bromide, nickel(II) iodide, nickel(II)sulfate, nickel(II) acetate, and mixtures thereof. The Ni(II) salt usedherein may be in any hydration state, for instance, nickel(II) nitrateincludes, without limitation, Ni(NO₃)₂, and Ni(NO₃)₂.6H₂O. In certainembodiments, a nickel salt having a different oxidation state, such as+3, may be used in addition to or in lieu of the Ni(II) salt. In apreferred embodiment, the Ni(II) salt is nickel(II) nitrate.

Non-limiting examples of the Co(II) salt include cobalt(II) nitrate,cobalt(II) chloride, cobalt(II) bromide, cobalt(II) iodide, cobalt(II)sulfate, cobalt(II) acetate, and mixtures thereof. The Co(II) salt usedherein may be in any hydration state, for instance, cobalt(II) nitrateincludes, without limitation, Co(NO₃)₂, and Co(NO₃)₂.6H₂O. In certainembodiments, a cobalt salt having a different oxidation state, such as+3, may be used in addition to or in lieu of the Co(II) salt.

Non-limiting examples of the Mn(II) salt include manganese(II) nitrate,manganese(II) sulfate, manganese(II) chloride, manganese(II) bromide,manganese(II) iodide, manganese(II) acetate, and mixtures thereof. TheMn(II) salt used herein may be in any hydration state, for instance,manganese(II) nitrate includes, without limitation, Mn(NO₃)₂, andMn(NO₃)₂.4H₂O. In certain embodiments, a manganese salt having adifferent oxidation state, such as +3, may be used in addition to or inlieu of the Mn(II) salt.

Preferably, the mesoporous silica nanoparticles may be initially driedto remove volatile impurities. The initial drying may be performed at atemperature of 80-200° C., 100-150° C., or about 120° C. for a period ofup to 48 hours, preferably up to 36 hours, or about 24 hours.

In one or more embodiments, mixing the Fe(III) salt and the M(II) salt(e.g. Cu(II), Co(II), Mn(II), and Ni(II)) with the mesoporous silicananoparticles to form a powdery mixture is conducted in neat(solvent-free) condition. Methods of agitating a powdery mixtureinclude, without limitation, using mortar and pestle, an agitator, avortexer, a rotary shaker, a magnetic stirrer, a centrifugal mixer, adual asymmetric centrifugal mixer, or an overhead stirrer. In oneembodiment, the powdery mixture is agitated by sonication in anultrasonic bath or with an ultrasonic probe. In another embodiment, themixture is mixed with a spatula. Alternatively, mixing the Fe(III) saltand the M(II) salt (e.g. Cu(II), Co(II), Mn(II), and Ni(II)) with themesoporous silica nanoparticles may be performed in the presence of asolvent such as water and alcohols (e.g. methanol, ethanol, n-propanol,i-propanol, n-butanol) to form a wet mixture. A powdery mixture may beprepared by precipitating and/or drying the wet mixture.

In one or more embodiments, a molar ratio of the M(II) salt (e.g.Cu(II), Co(II), Mn(II), and Ni(II)) to the Fe(III) salt is in a range of1:0.5 to 1:4, preferably 1:0.7 to 1:3, more preferably 1:0.9 to 1:2, orabout 1:1.

The powdery mixture may be calcined in air within a furnace or oven at atemperature of 600-1,100° C., preferably 700-1,000° C., preferably750-900° C., preferably 800-850° C., though in some embodiments, thepowdery mixture may be heated at a temperature lower than 600° C. orhigher than 1,100° C. In some embodiments, the powdery mixture may notbe heated in air, but oxygen-enriched air, an inert gas, or a vacuum.The powdery mixture may be maintained at the calcining temperature for1-12 hours, 2-10 hours, 4-8 hours, or about 6 hours. Calcining thepowdery mixture produces the nanoformulation.

In another embodiment, it is equally envisaged that the method ofproducing the nanoformulation may be adapted to other means ofdispersing and impregnating the spinel ferrite on the mesoporous silicananoparticles. Exemplary other means include, but are not limited to,isomorphous substitution, enforced impregnation, vapor-fed flamesynthesis, flame spray pyrolysis, sputter deposition, atomic layerdeposition, and chemical vapor deposition.

The method further involves mixing the nanoformulation and a platinumcomplex in an aqueous solution, thereby forming the nanotherapeutic. Theplatinum complex is the same as previously described.

In one or more embodiments, the aqueous solution is an aqueous solutioncomprising an alkali salt, an alkaline earth salt, and/or an ammoniumsalt. Non-limiting examples of salt present in the aqueous solutioninclude sodium or potassium chloride, sodium or potassium bromide,sodium or potassium bicarbonate, sodium or potassium sulfate, sodium orpotassium carbonate, and ammonium chloride. Preferably, the aqueoussolution is normal saline solution (NSS) that contains sodium chlorideat a concentration of 0.8-1.0 wt %, preferably 0.85-0.95 wt %, or about0.9 wt % relative to a total volume of the solution.

In one embodiment, the platinum complex is present in the aqueoussolution at a concentration of 0.1-30 g/L relative to a total volume ofthe aqueous solution, preferably 0.5-25 g/L, preferably 1-20 g/L,preferably 1.5-15 g/L, preferably 2-10 g/L, preferably 2.5-5 g/L, orabout 3 g/L relative to a total volume of the aqueous solution. In arelated embodiment, the nanoformulation is present in the aqueoussolution at a concentration of 2-600 g/L relative to a total volume ofthe aqueous solution, preferably 5-500 g/L, preferably 10-400 g/L,preferably 20-300 g/L, preferably 30-200 g/L, preferably 40-100 g/L,preferably 50-75 g/L, or about 60 g/L relative to a total volume of theaqueous solution.

Mixing the nanoformulation and the platinum complex in the aqueoussolution may be performed at a temperature of −15-15° C., preferably−10-10° C., preferably −4-4° C. for 2-48 hours, 6-24 hours, or 12-18hours. Alternatively, the mixing may continue until no net transferbetween the platinum complex and the nanoformulation is observed. Thenet transfer may be determined by monitoring the mixing process usingUV-vis, and/or FT-IR spectroscopies. In a preferred embodiment, themixing is conducted in the absence of light (e.g. keeping the mixture inthe dark). The nanotherapeutic containing the platinum complexencapsulated in the pores of the nanoformulation may be collected viafiltration, washed with a solution such as water and normal salinesolution, and dried in air or in a vacuum.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism, does not abrogate the biological activity and properties ofthe administered active ingredient, and/or does not interact in adeleterious manner with the other components of the composition in whichit is contained. The term “carrier” encompasses any excipient, binder,diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or othermaterial well known in the art for use in pharmaceutical formulations.The choice of a carrier for use in a composition will depend upon theintended route of administration for the composition. The preparation ofpharmaceutically acceptable carriers and formulations containing thesematerials is described in, e.g. Remington's Pharmaceutical Sciences,21st Edition, ed. University of the Sciences in Philadelphia,Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which isincorporated herein by reference in its entirety).

In one or more embodiments, the nanotherapeutic further comprises one ormore pharmaceutically acceptable carriers and/or excipients selectedfrom the group consisting of a buffer, an inorganic salt, a fatty acid,a vegetable oil, a synthetic fatty ester, a surfactant, and a polymer.

In a preferred embodiment, the nanotherapeutic comprises a biocompatiblepolymer, such as chitosan. Chitosan is a polysaccharide copolymer ofN-acetyl-D-glucosamine and D-glucosamine, obtained by the alkalinedeacetylation of chitin shells obtained from crustaceans, such asshrimps and crabs. The chitosan may or may not be quaternized chitosan.Derivatives of chitosan, such as chitosan oligosaccharide lactate,trimethylchitosan, and glycol chitosan, which have a higher solubilityin water than the unmodified chitosan may be preferred. The chitosan orderivative thereof in the nanotherapeutic may have a weight averagemolecular weight ranging from 5-100 kDa, preferably 10-80 kDa, morepreferably 20-50 kDa. The weight average molecular weight may bemeasured by gel permeation chromatography. Other exemplary polymersinclude, without limitation, polylactides, polyglycolides,polycaprolactones, polyanhydrides, polyurethanes, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), poly(maleic anhydride), apolyvinyl alcohols, and copolymers, terpolymers, or combinations ormixtures therein. The copolymer/terpolymer may be a randomcopolymer/terpolymer, or a block copolymer/terpolymer.

Exemplary buffers include, but are not limited to, phosphate buffers,citrate buffer, acetate buffers, borate buffers, carbonate buffers,bicarbonate buffers, and buffers with other organic acids and salts.

Exemplary inorganic salts include, but are not limited to, calciumcarbonate, calcium phosphate, disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, zinc oxide, zinc sulfate, andmagnesium trisilicate.

Exemplary fatty acids include, but are not limited to, an omega-3 fattyacid (e.g., linolenic acid, docosahexaenoic acid, eicosapentaenoic acid)and an omega-6 fatty acid (e.g., linoleic acid, eicosadienoic acid,arachidonic acid). Other fatty acids, such as oleic acid, palmitoleicacid, palmitic acid, stearic acid, and myristic acid, may be included.

Exemplary vegetable oils include, but are not limited to, avocado oil,olive oil, palm oil, coconut oil, rapeseed oil, soybean oil, corn oil,sunflower oil, cottonseed oil, and peanut oil, grape seed oil, hazelnutoil, linseed oil, rice bran oil, safflower oil, sesame oil, brazil nutoil, carapa oil, passion fruit oil, and cocoa butter.

Exemplary synthetic fatty esters include, without limitation, methyl,ethyl, isopropyl and butyl esters of fatty acids (e.g., isopropylpalmitate, glyceryl stearate, ethyl oleate, isopropyl myristate,isopropyl isostearate, diisopropyl sebacate, ethyl stearate, di-n-butyladipate, dipropylene glycol pelargonate), C₁₂-C₁₆ fatty alcohol lactates(e.g., cetyl lactate and lauryl lactate), propylene dipelargonate,2-ethylhexyl isononoate, 2-ethylhexyl stearate, isopropyl lanolate,2-ethylhexyl salicylate, cetyl myristate, oleyl myristate, oleylstearate, oleyl oleate, hexyl laurate, isohexyl laurate, propyleneglycol fatty ester, and polyoxyethylene sorbitan fatty ester. As usedherein, the term “propylene glycol fatty ester” refers to a monoether ordiester, or mixtures thereof, formed between propylene glycol orpolypropylene glycol and a fatty acid. The term “polyoxyethylenesorbitan fatty ester” denotes oleate esters of sorbitol and itsanhydrides, typically copolymerized with ethylene oxide.

Surfactants may act as detergents, wetting agents, emulsifiers, foamingagents, and dispersants. Surfactants that may be present in thecompositions of the present disclosure include zwitterionic (amphoteric)surfactants, e.g., phosphatidylcholine, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),anionic surfactants, e.g., sodium lauryl sulfate, sodium octanesulfonate, sodium decane sulfonate, and sodium dodecane sulfonate,non-ionic surfactants, e.g., sorbitan monolaurate, sorbitanmonopalmitate, sorbitan trioleate, polysorbates such as polysorbate 20(Tween 20), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80),cationic surfactants, e.g., decyltrimethylammonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,tetradecyltrimethyl-ammonium chloride, and dodecylammonium chloride, andcombinations thereof.

The term “active ingredient”, as used herein, refers to an ingredient inthe nanotherapeutic that is biologically active, for example, theplatinum complex. In some embodiments, other active ingredients inaddition to the platinum complex may be incorporated into thenanotherapeutic.

In one embodiment, the nanotherapeutic includes a second activeingredient, such as a chemotherapeutic agent or an anticancer agent thatis structurally different from the platinum complex, for the treatmentor prevention of neoplasm of tumor or cancer cell division, growth,proliferation and/or metastasis in the subject; induction of death orapoptosis of tumor and/or cancer cells; and/or any other forms ofproliferative disorder.

The second anticancer agent may be at least one of a mitotic inhibitor;an alkylating agent; an antimetabolite; a cell cycle inhibitor; anenzyme; a topoisomerase inhibitor; a biological response modifier; ananti-hormone; an antiangiogenic agent such as MMP-2, MMP-9 and COX-2inhibitor; an anti-androgen; a substituted urea such as hydroxyurea; amethylhydrazine derivative; an adrenocortical suppressant, e.g.,mitotane, aminoglutethimide; a hormone and/or hormone antagonist such asthe adrenocorticosteriods (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate), an estrogen (e.g., diethylstilbestrol);an antiestrogen such as tamoxifen; androgen, e.g., testosteronepropionate; and an aromatase inhibitor, such as anastrozole, andAROMASIN (exemestane).

Exemplary anticancer agents include, but are not limited to, alkylatingantineoplastic agents including busulfan, carmustine, chlorambucil,cyclophosphamide, cyclophosphamide, dacarbazine, ifosfamide, lomustine,mechlorethamine, melphalan, mercaptopurine, procarbazine;antimetabolites including cladribine, cytarabine, fludarabine,gemcitabine, pentostatin, 5-fluorouracil, clofarabine, capecitabine,methotrexate, thioguanine; anti-microtubule agents including etoposide,vinblastine, vincristine, teniposide, docetaxel, paclitaxel,vinorelbine, vindesine; cytotoxic antibiotics including daunorubicin,doxorubicin, idarubicin, mitomycin, actinomycin, epirubicin;topoisomerase inhibitors including irinotecan, mitoxantrone, topotecan,and mixtures thereof.

In preferred embodiments, the nanotherapeutic having the activeingredient (e.g. the platinum complex) has pH-dependent controlledrelease. A pH-dependent controlled release of the active ingredientmeans that the amount of active ingredient which is released ordissolved in the medium at a certain time interval varies significantlywith different pH values. For example, when the nanotherapeutic isexposed in a medium of pH 5 (e.g. acidic tumor microenvironment), atleast 35 wt % of the loaded active ingredient is cumulatively releasedfrom the nanotherapeutic within 24-80 hours, 36-72 hours, or 48-60hours, preferably at least 40 wt %, preferably at least 50 wt %,preferably at least 60 wt %, preferably at least 70 wt %, preferably atleast 80 wt %, preferably at least 85 wt %, preferably at least 90 wt %,preferably at least 95 wt %, preferably at least 97 wt %, preferably atleast 99 wt % of the loaded active ingredient is cumulatively releasedfrom the nanotherapeutic within 24-80 hours, 36-72 hours, or 48-60 hours(see FIGS. 8 and 9 ). While at higher pH levels, for instance atphysiological pH 7.4, less than 30 wt % of the loaded active ingredientis cumulatively released from the nanotherapeutic within 24-80 hours,36-72 hours, or 48-60 hours, preferably less than 25 wt %, preferablyless than 20 wt %, preferably less than 15 wt %, preferably less than 10wt %, preferably less than 5 wt %, preferably less than 2 wt %,preferably less than 1 wt % of the loaded active ingredient iscumulatively released from the nanotherapeutic within 24-72 hours, 36-60hours, or 42-54 hours.

In one embodiment, the nanotherapeutic having the active ingredient(e.g. the platinum complex) has sustained-release. Sustained-releaserefers to a release of an active ingredient from a composition or dosageform in which the active ingredient is released over an extended periodof time. In one embodiment, sustained-release occurs when there isdissolution of an active ingredient within 0.5-80 hours, preferablywithin 1-72 hours, more preferably within 24-48 hours after beingswallowed. In another embodiment, sustained-release occurs when there isdissolution of an active ingredient within 0.5-80 hours, preferablywithin 1-72 hours, more preferably within 24-48 hours after entering theintestine. In another embodiment, sustained-release results insubstantially complete dissolution after at least 1 hour, preferablyafter at least 24 hours, more preferably after at least 48 hoursfollowing administration. In another embodiment, sustained-releaseresults in substantially complete dissolution after at least 1 hour,preferably after at least 24 hours, more preferably after at least 48hours following oral administration. In another embodiment,sustained-release results in substantially complete dissolution after atleast 1 hour, preferably after at least 24 hours, more preferably afterat least 48 hours following rectal administration.

On the contrary, immediate release refers to the release of an activeingredient substantially immediately upon administration. In anotherembodiment, immediate release occurs when there is dissolution of anactive ingredient within 1-20 minutes after administration. Dissolutioncan be of all or less than all (e.g. about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, about 99.5%, 99.9%, or 99.99%) ofthe active ingredient. In another embodiment, immediate release resultsin complete or less than complete dissolution within about 1 hourfollowing administration. Dissolution can be in a subject's stomachand/or intestine. In one embodiment, immediate release results indissolution of an active ingredient within 1-20 minutes after enteringthe stomach. For example, dissolution of 100% of an active ingredientcan occur in the prescribed time. In another embodiment, immediaterelease results in complete or less than complete dissolution withinabout 1 hour following rectal administration. In some embodiments,immediate release is through inhalation, such that dissolution occurs ina subject's lungs. In one embodiment, the nanotherapeutic having theactive ingredient (e.g. the platinum complex) has immediate release.

In some embodiments, the active ingredient released from thenanotherapeutic, e.g. the platinum complex, provides utility as ananticancer agent in reducing the viability of cancer cells derived fromhuman cancer cell lines including, but not limited to, breast cancercell lines (e.g. MCF-7, and SK-BR-3), ovarian cancer cell lines (e.g.NCI-ADR/RES, OVCAR-03), colon cancer cell lines (e.g. HCT-116, HT-29),liver cancer cell lines (e.g. HepG2), lung cancer cell lines (e.g. A549,NCI-H460), brain tumor cell lines (e.g. U251), prostate cancer celllines (e.g. PC-3), renal cancer cell lines (e.g. 786-0), and melanomacell lines (e.g. UACC-62).

In some embodiments, the ability of the nanotherapeutic to reduce theviability of cancer cells may be determined by contacting thenanotherapeutic with the cancer cells and then performing cell viabilityassays. Methods of such assays include, but are not limited to, ATPtest, Calcein AM assay, clonogenic assay, ethidium homodimer assay,Evans blue assay, fluorescein diacetate hydrolysis/propidium iodidestaining assay, flow cytometry, Formazan-based assays (MTT, XTT), greenfluorescent protein assay, lactate dehydrogenase (LDH) assay, methylviolet assay, propidium iodide assay, Resazurin assay, trypan blueassay, and TUNEL assay. In a preferred embodiment, a MTT assay is used.

According to another aspect, the present disclosure relates to a methodfor treating a proliferative disorder. The method involves administeringthe nanotherapeutic of the first aspect to a subject in need of therapy.

As used herein, the terms “treat”, “treatment”, and “treating” in thecontext of the administration of a therapy to a subject in need thereofrefer to the reduction or inhibition of the progression and/or durationof a disease (e.g. cancer), the reduction or amelioration of theseverity of the disease, and/or the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies.“Treating” or “treatment” of the disease includes preventing the diseasefrom occurring in a subject that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), ameliorating the disease, providing relief from thesymptoms or side-effects of the disease (including palliativetreatment), and relieving the disease (causing regression of thedisease). With regard to the disease, these terms simply mean that oneor more of the symptoms of the disease will be reduced. Such terms mayrefer to one, two, three, or more results following the administrationof one, two, three, or more therapies: (1) a stabilization, reduction(e.g. by more than 10%, 20%, 30%, 40%, 50%, preferably by more than 60%of the population of cancer cells and/or tumor size beforeadministration), or elimination of the cancer cells, (2) inhibitingcancerous cell division and/or cancerous cell proliferation, (3)relieving to some extent (or, preferably, eliminating) one or moresymptoms associated with a pathology related to or caused in part byunregulated or aberrant cellular division, (4) an increase indisease-free, relapse-free, progression-free, and/or overall survival,duration, or rate, (5) a decrease in hospitalization rate, (6) adecrease in hospitalization length, (7) eradication, removal, or controlof primary, regional and/or metastatic cancer, (8) a stabilization orreduction (e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,preferably at least 80% relative to the initial growth rate) in thegrowth of a tumor or neoplasm, (9) an impairment in the formation of atumor, (10) a reduction in mortality, (11) an increase in the responserate, the durability of response, or number of patients who respond orare in remission, (12) the size of the tumor is maintained and does notincrease or increases by less than 10%, preferably less than 5%,preferably less than 4%, preferably less than 2%, (13) a decrease in theneed for surgery (e.g. colectomy, mastectomy), and (14) preventing orreducing (e.g. by more than 10%, more than 30%, preferably by more than60% of the population of metastasized cancer cells beforeadministration) the metastasis of cancer cells.

The term “subject” and “patient” are used interchangeably. As usedherein, they refer to any subject for whom or which therapy, includingwith the compositions according to the present disclosure is desired. Inmost embodiments, the subject is a mammal, including but not limited toa human, a non-human primate such as a chimpanzee, a domestic livestocksuch as a cattle, a horse, a swine, a pet animal such as a dog, a cat,and a rabbit, and a laboratory subject such as a rodent, e.g. a rat, amouse, and a guinea pig. In preferred embodiments, the subject is ahuman.

In one or more embodiments, the proliferative disorder is cancer. Insome embodiments, the disclosed method of the current aspect is fortreating cancer of the breast, ovary, cervix, testicle, colon, bladder,lung, blood, brain, rectum, pancreas, skin, prostate gland, stomach,spleen, liver, kidney, head, neck, bone, bone marrow, thyroid gland, orcentral nervous system. In a preferred embodiment, the cancer is atleast one selected from the group consisting of breast cancer, ovariancancer, cervical cancer, testicular cancer, colon cancer, bladdercancer, and lung cancer. In a more preferred embodiment, the cancer isbreast cancer.

As used herein, a subject in need of therapy includes a subject alreadywith the disease, a subject which does not yet experience or exhibitsymptoms of the disease, and a subject predisposed to the disease. Inpreferred embodiments, the subject is a person who is predisposed tocancer, e.g. a person with a family history of cancer. For example,white women or a person with (i) certain inherited genes (e.g. mutatedBRCA1, BRCA2, ATM, TP53, CHEK2, PTEN, CDH1, STK11, and PALB2), (ii)radiation occurred to one's chest, and/or (iii) exposure todiethylstilbestrol (DES) are at a higher risk of contracting breastcancer. People who (i) had inflammatory bowel disease, or a geneticsyndrome such as familial adenomatous polyposis (FAP) and hereditarynon-polyposis colorectal cancer (Lynch syndrome), and/or (ii) consumes alow-fiber and high-fat diet are at a higher risk of contracting coloncancer. People who (i) smoke or regularly breathe in second-hand smoke,(ii) exposed to carcinogens such as asbestos, radioactive substances(e.g., uranium, radon), and/or (iii) inhaled chemicals or minerals(e.g., arsenic, beryllium, cadmium, silica, vinyl chloride, nickelcompounds, chromium compounds, coal products, mustard gas, andchloromethyl ethers) are at a higher risk of contracting lung cancer. Aperson with (i) chronic infection with the hepatitis B virus (HBV) orhepatitis C virus (HCV), (ii) cirrhosis of the liver, (iii) nonalcoholicfatty liver disease, and/or (iv) exposure to aflatoxins is at a higherrisk of contracting liver cancer.

Other non-cancerous proliferative disorders that may also be treated bythe currently disclosed nanotherapeutic include, without limitation,atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonaryfibrosis, scleroderma, cirrhosis of the liver, lymphoproliferativedisorder, other disorders characterized by epidermal cell proliferationsuch as verruca (warts), and dermatitis, and benign proliferative breastdisease such as ductal hyperplasia, lobular hyperplasia, and papillomas.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to the methods that may be used to enable deliveryof the active ingredient and/or the composition to the desired site ofbiological action. Routes or modes of administration are as set forthherein. These methods include, but are not limited to, oral routes,intraduodenal routes, parenteral injection (including intravenous,subcutaneous, intraperitoneal, intramuscular, intravascular, orinfusion), topical and rectal administration. Those of ordinary skill inthe art are familiar with administration techniques that can be employedwith the nanotherapeutics and methods described herein.

Cisplatin is widely prescribed in chemotherapy medications for thetreatment of breast, ovarian, cervical, testicular, lung, bladder, headand neck cancers. In one or more embodiments, the platinum complexloaded in the nanotherapeutic is preferably cisplatin and derivatives,e.g. carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate,phenanthriplatin, picoplatin, satraplatin, lobaplatin, heptaplatin,dicycloplatin, other platinum-based antineoplastic drugs, and mixturesthereof. Preferably, the platinum complex is cisplatin.

The dosage amount and treatment duration are dependent on factors, suchas bioavailability of a drug, administration mode, toxicity of a drug,gender, age, lifestyle, body weight, the use of other drugs and dietarysupplements, the disease stage, tolerance and resistance of the body tothe administered drug, etc., and then determined and adjustedaccordingly. The terms “effective amount”, “therapeutically effectiveamount”, or “pharmaceutically effective amount” refer to that amount ofthe active ingredient being administered which will relieve to someextent one or more of the symptoms of the disease being treated. Theresult can be a reduction and/or alleviation of the signs, symptoms, orcauses of a disease, or any other desired alteration of a biologicalsystem. An appropriate “effective amount” may differ from one individualto another. An appropriate “effective amount” in any individual case maybe determined using techniques, such as a dose escalation study.

In one or more embodiments, an effective amount of the nanotherapeuticin a range of 0.5-800 mg/kg, preferably 1-500 mg/kg, more preferably10-100 mg/kg is administered per body weight of the subject. However, incertain embodiments, the effective amount of the nanotherapeutic is lessthan 0.5 mg/kg or greater than 800 mg/kg per body weight of the subject.

In treating certain cancers, the best approach is often a combination ofsurgery, radiotherapy, and/or chemotherapy. Therefore, in at least oneembodiment, the nanotherapeutic is employed in conjunction withradiotherapy. In another embodiment, the nanotherapeutic is employedwith surgery. The radiotherapy and/or surgery may be before or after thenanotherapeutic is administered.

A treatment method may comprise administering the nanotherapeutic of thecurrent disclosure in any of its embodiments as a single dose ormultiple individual divided doses. In some embodiments, thenanotherapeutic is administered at various dosages (e.g. a first dosewith an effective amount of 200 mg/kg and a second dose with aneffective amount of 50 mg/kg). In some embodiments, the interval of timebetween the administration of the nanotherapeutic and the administrationof one or more additional therapies may be about 1-5 minutes, 1-30minutes, 30 minutes to 60 minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 1 year, 2 years, or any periodof time in between. Preferably, the nanotherapeutic is administered oncedaily for at least 2 days, at least 5 days, at least 6 days, or at least7 days. In certain embodiments, the nanotherapeutic and one or moreadditional therapies are administered less than 1 day, less than 1 week,less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 1month, less than 2 months, less than 3 months, less than 6 months, lessthan 1 year, less than 2 years, or less than 5 years apart.

The methods for treating cancer and other proliferative disordersdescribed herein inhibit, remove, eradicate, reduce, regress, diminish,arrest or stabilize a cancerous tumor, including at least one of thetumor growth, tumor cell viability, tumor cell division andproliferation, tumor metabolism, blood flow to the tumor and metastasisof the tumor. In some embodiments, the size of a tumor, whether byvolume, weight or diameter, is reduced after the treatment by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100%, relative to the tumor size before treatment. In otherembodiments, the size of a tumor after treatment does not reduce but ismaintained the same as the tumor size before treatment. Methods ofassessing tumor size include, but are not limited to, CT scan, MRI,DCE-MRI and PET scan.

In most embodiments, the method further comprises measuring aconcentration of a biomarker and/or detecting a mutation in a biomarkerbefore and/or after the nanotherapeutic of the present disclosure isadministered. As used herein, the term “biomarker” refers to acharacteristic that is objectively measured and evaluated as anindicator of normal biological processes, pathogenic processes orpharmacological responses to a therapeutic intervention. Generic cancerbiomarkers include circulating tumor DNA (ctDNA) and circulating tumorcells (CTC).

Exemplary cancer biomarkers for breast cancer include, withoutlimitation, BRCA1, BRCA2, HER-2, estrogen receptor, progesteronereceptor, cancer antigen 15-3, cancer antigen 27.29, carcinoembryonicantigen, Ki67, cyclin D1, cyclin E, and ERβ. Specifically, potentiallypredictive cancer biomarkers include, without limitation, mutations ingenes BRCA1 and BRCA2 for breast cancer. Exemplary biomarkers for coloncancer include, without limitation, carcinoembryonic antigen (CEA),carbohydrate antigen 242 (CA 242), CA 195, CA 19-9, MSI, and 18qLOH.Exemplary biomarkers for lung cancer include, without limitation, CA125, CA 15-3, EGF receptor, anaplastic lymphoma kinase gene, MET, ROS-1,and KRAS. Exemplary biomarkers for liver cancer include, withoutlimitation, alpha-fetoprotein (AFP), AFP-L3, des-γ-carboxyprothrombin(DCP), GPC3, GP73, cytokeratin 19 (CK 19), osteopontin, IL-6, midkine(MDK), and Annexin A2.

Potentially predictive cancer biomarkers include, without limitation,overexpressions of CEA, NSE, CYFRA-21-1, CA-125, and CA-199 for lungcancer, overexpressions of TYMS, mutations in genes p53 and KRAS forcolon cancer, and high concentration levels of AFP, and overexpressionsof HSP90α for liver cancer.

The mutation in the biomarker may be detected by procedures such asrestriction fragment length polymorphism (RFLP), polymerase chainreaction (PCR) assay, multiplex ligation-dependent probe amplification(MLPA), denaturing gradient gel electrophoresis (DGGE), single-strandconformation polymorphism (SSCP), hetero-duplex analysis, proteintruncation test (PTT), and oligonucleotide ligation assay (OLA). Theprocedures to detect the mutation are well-known to those of ordinaryskill in the art.

The term “sample” used herein refers to any biological sample obtainedfrom the subject in need of therapy including a single cell, multiplecells, fragments of cells, a tissue sample, and/or body fluid.Specifically, the biological sample may include red blood cells, whiteblood cells, platelets, hepatocytes, epithelial cells, endothelialcells, a skin biopsy, a mucosa biopsy, an aliquot of urine, saliva,whole blood, serum, plasma, lymph. In some embodiments, the biologicalsample is taken from a tumor.

The concentration level of the cancer biomarker in a sample may bemeasured by an assay, for example an immunoassay. Typical immunoassaymethods include, without limitation, enzyme-linked immunosorbent assay(ELISA), enzyme-linked immunospot assay (ELISPOT), Western blotting,immunohistochemistry (IHC), immunocytochemistry, immunostaining, andmultiple reaction monitoring (MRM) based mass spectrometric immunoassay.The protocol for measuring the concentration of the biomarker and/ordetecting the mutation in the biomarker is known to those of ordinaryskill, for example by performing the steps outlined in the commerciallyavailable assay kit sold by Sigma-Aldrich, Thermo Fisher Scientific, R &D Systems, ZeptoMetrix Inc., Cayman Inc., Abcam, Trevigen, DojindoMolecular Technologies, Biovision, and Enzo Life Sciences.

In some embodiments, a concentration of the biomarker is measured beforeand after the administration. When the concentration of the biomarker ismaintained, the method may further comprise increasing the effectiveamount of the nanotherapeutic by at least 5%, at least 10%, or at least30%, up to 50%, up to 60%, or up to 80% of an initial effective amountthat is in a range of 0.5-800 mg/kg per body weight of the subject. Theincreased effective amount may be in a range of 0.525-1,440 mg/kg,preferably 5-1,000 mg/kg, more preferably 50-500 mg/kg. The subject maybe administered with the increased dosage for a longer period (e.g. 1week more, 2 weeks more, or 2 months more) than the duration prescribedwith the initial effective amount.

In some embodiments, the mutation in the biomarker is detected beforeadministering the composition to identify subjects predisposed to thedisease. Alternatively, the biomarkers are measured/detected after eachadministration. For example, the measurement may be 30-60 minutes, 1-2hours, 2-12 hours, 12-24 hours, 1-2 days, 1-15 weeks, 15-20 weeks, 20-30weeks, 30-40 weeks, 40-50 weeks, 1 year, 2 years, or any period of timein between after the administration.

In some embodiments, the administration is stopped once the subject istreated.

The examples below are intended to further illustrate protocols forpreparing, characterizing the nanotherapeutic, and uses thereof, and arenot intended to limit the scope of the claims.

Example 1

Preparation of HYPS Nanoformulations

The support HYPS was purchased from Superior silica, USA.

The support HYPS was predried at 120° C. for 24 h before the preparationof nanoformulations.

For 30% CoFe₂O₄; 0.74 g of cobalt nitrate hexahydrate, 1.03 g of ironnitrate nonahydrate was taken and mixed with 1.4 g of HYPS using mortarand pestle and then calcined at 850° C. for 6 h.

For 30% NiFe₂O₄; 0.74 g of nickel nitrate hexahydrate, 1.03 g of ironnitrate nonahydrate was taken and mixed with 1.4 g of HYPS using mortarpistol and then calcined at 850° C. for 6 h.

For 30% CuFe₂O₄; 0.61 g of copper nitrate trihydrate, 1.01 g of ironnitrate nonahydrate was taken and mixed with 1.4 g of HYPS using mortarpistol and then calcined at 850° C. for 6 h.

For 30% MnFe₂O₄; 0.64 g of manganese nitrate tetrahydrate, 1.05 g ofiron nitrate nonahydrate was taken and mixed with 1.4 g of HYPS usingmortar pistol and then calcined at 850° C. for 6 h.

Example 2

Preparation of the Nanotherapeutics

The cisplatin loading was carried out by dissolving an appropriateamount of cisplatin (30 mg) in normal saline solution (NSS) followingour previously published literature [Jermy B R, Acharya S, RavinayagamV, Alghamdi H S, Akhtar S, Basuwaidan R S (2018) Hierarchicalmesosilicalite nanoformulation integrated with cisplatin exhibitstarget-specific efficient anticancer activity. Appl Nanosci 8,1205-1220, incorporated herein by reference in its entirety] to yield30% CuFe₂O₄/HYPS-cisplatin. Chitosan wrapped CuFe₂O₄/HYPS was preparedas follows. 0.6 wt % chitosan solution was prepared using 10% (v/v)acetic acid solution via overnight stirring. The initial pH of thechitosan solution was 2.77. Then, 1 M NaOH solution was added to thechitosan solution drop wise to increase the pH to 6.4. 1 g cisplatinloaded CuFe₂O₄/HYPS was added to the solution and the mixture was keptunder stirring for 24 hours. The pH was increased to 7 afterwards. Themixture was kept under stirring for another 24 hours, then centrifuged,washed and dried under vacuum for 48 hours at 37° C.

Example 3

Catalyst Characterization

X-ray diffraction patterns of the SPIONs/silica nanoformulations wereanalyzed using bench top Rigaku Multiplex system (Rigaku, Japan).Textural characteristics involving surface area and pore sizedistributions of the parent support and nanoformulations were measuredusing an ASAP-2020 plus (Micromeritics, USA). Cisplatin functionalgroups were identified using FT-IR spectroscopy equipped with attenuatedtotal reflectance (ATR) (Perkin Elmer, USA). Surface features of thesynthesized materials were characterized by scanning electron microscopy(SEM) and transmission electron microscopy (TEM). For SEM (FEI, InspectS50, Czech Republic), the prepared powder was dispersed onto doublesided tape holder and examined under 20 kV. Depending on the morphology,different magnifications were chosen to capture the representativefeatures of the specimens. For SPIONs/S-16 specimen, the micrographswere taken at representative magnifications of 10,000 and 20,000×, while20,000 and 50,000× were applied for SPIONs/HYPS and SPIONs/MSU-Fspecimens. TEM samples were prepared by dispersing a small amount ofsample in ethanol and deposited onto TEM grids. The grids were examinedby a TEM instrument (FEI, Inspect S50, Czech Republic) at a workingvoltage of 80 kV. Several images were acquired to measure the particlesize and calculate an average size. The particle size was measured usingGatan digital micrograph software. The results are displayed in the formof size histogram for each prepared sample.

Example 4

Drug Adsorption Study

In cisplatin adsorption study, spinel ferrite/silica nanoformulations(600 mg) were independently mixed with 30 mg of cisplatin in 10 mL ofsaline solution under ice cooled dark environment. After stirringovernight, the solution mixture was filtered, and washed with 15 mLnormal saline solution. Then the amount of adsorbed cisplatin wascalculated via UV-visible spectroscopy using the wavelength at 208 nm.

Example 5

Drug Release Study

The cumulative cisplatin release was studied using differentnanoformulations involving HYPS (i.e. nanotherapeutics). Cellulosemembrane dialysis tubing was activated, and drug delivery analysis wasperformed by immersing the bag containing 30 mg of nanoformulations in50 mL of phosphate buffered saline (PBS) at pH 5.6. The releasecondition was performed under stabilized temperature of 37° C. At aspecified time interval, a 10 mL solution was removed and analyzed usingUV-visible spectroscopy.

Example 6

In-Vitro Study on MCF-7 Cells

In this disclosure, the antitumor effect of nanotherapeutics includingcisplatin loaded 30 wt % CuFe₂O₄/HYPS and 30 wt % CuFe₂O₄/HYPS/chitosanwas tested on human mammary adenocarcinoma cell line, MCF-7. Cells weremaintained in DMEM (Dulbecco's Modified Eagle Medium) (Gibco, lifetechnologies) supplemented with 10% heat inactivated fetal bovine serum(HI-FBS) (Gibco, life technology), 1% Penicillin Streptomycin (100×—Gibco, life technology), and 1% MEM NEAA (MEM non-essential amino acids)(100×— Gibco, life technology). Cells were kept in a humidifiedincubator at 37° C. with 5% CO₂. For the experimental setup, MCF-7 cellswere seeded on a 96-well plate at a density of 20,000 cells/well. On thenext day, cells were shifted to the starve media (0.5% HI-FBS containingmedia) for 24 h before treatment.

Treatment Conditions:

Group A—CuFe₂O4

Group B— Nanomaterial (i.e. mesoporous silica)

Group C— Nanomaterial+CuFe₂O₄ (i.e. nanoformulation)

Group D—Cisplatin

Group E—Cisplatin+Nanomaterial+CuFe₂O₄ (i.e. nanotherapeutic)

Group F— Cisplatin+Nanomaterial+CuFe₂O₄+Chitosan

Cells were treated for 48 hours with the following conditions: group A(CuFe₂O₄), group B (nanomaterial), group C (nanomaterial+CuFe₂O₄), groupD (cisplatin), group E (cisplatin+nanomaterial+CuFe₂O₄), and group F(cisplatin+nanomaterial+CuFe₂O₄+chitosan). For groups B, C, E, and F,treatment concentrations were as follows: 0.025, 0.05, 0.1, and 0.5mg/mL. To accurately reflect the concentration of CuFe₂O₄ (group A), andcisplatin (group D) that is encapsulated within these nanoparticles, thedrug loading experiments were used to calculate the actual concentrationof each in the other groups. Therefore, treatment concentrations forgroup A were as follows: 0.0084, 0.0168, 0.0336, and 0.168 mg/mL.Treatment concentrations used in this experiment for group D were asfollows: 0.001125, 0.00225, 0.0045, 0.0225 mg/mL.

Example 7

Cell Viability—MTT Assay

The viability of cells was tested using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. It is based on the ability to reduce MTT relative to formazancrystals. The assay was performed using previously published protocols[Mosmann T. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. ImmunolMethods. 1983 Dec. 16; 65(1-2): 55-63]. Briefly, MTT (Sigma-Aldrich) wasdissolved in PBS at a concentration of 5 mg/mL. Working solution of MTTwas prepared at a final concentration of 0.5 mg/mL (10 μL of stockMTT+90 μL 1×PBS/well). The 96 wells plate was washed twice with 1×PBSand 100 μL of MTT working solution was dispended in all wells. An MTTbackground control was included, in which MTT working solution was addedto empty wells (no cells). The plate was incubated for three hours at37° C., followed by the addition of 100 μL of acidified isopropanolsolubilizing solution (0.04 N HCl isopropanol). The change in colorintensity was measured at 570 nm wavelength using SYNERGY-neo2 BioTekELISA reader. Each condition was performed in triplicates. The readingof each triplicate was averaged and subtracted from the averaged MTTbackground control reading. Each condition was compared to the control(no treatment) wells. The following equation was used to calculate the %of cell viability:

${\%{Cell}{Viability}} = {\frac{ave{raged}{sample}{read}}{{averaged}{control}{read}} \times 100}$

Example 8

Statistical Analysis

Cell viability assay data represent four independent experiments.Statistical analysis was performed using Prism 7 software (GraphPad, LaJolla, Calif.). Analysis was performed using two-way ANOVA withDunnett's post hoc test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001versus control.

Example 9

Results and Discussions: Nanoformulations

FIG. 1 shows the XRD diffraction patterns of 30% CuFe₂O₄ loaded on HYPSusing dry impregnation technique. The presence of a characteristicsbroad peak due to amorphous siliceous framework was observed between15-30°. In case of metal oxides, the diffraction patterns correlate withthe cubic phase of copper ferrite (JCPDS 77-0010). However, the presenceof relatively less crystalline CuFe₂O₄ nanoparticles demonstrated byweak peaks and increased broadness clearly indicates the presence ofsmall nanosized nanoparticles. Such trend indicates the lack ofcrystallization at spherical nanopores of HYPS (FIG. 1 ).

As shown in FIG. 2 , the intensity of spinel increases with increasingcopper content. This shows that the occupation of copper in octahedralpositions may improve the crystallinity.

XRD diffraction patterns of CuFe₂O₄/HYPS calcined at differenttemperatures ranging between 700-900° C. are shown in FIG. 3 . Theintensity of CuFe₂O₄ main intensity peak at 35° tends to increase withincreasing calcination temperature. However, at lower calcinationtemperatures pure spinel components dominate, while increasingtemperature tends to produce mixed phase along with copper iron oxidethough in a minor amount. The pattern shows spinel crystal growth withincreasing temperature, while presence of broad peak corresponding tosilica even at high temperature of 900° C. shows the preservation ofamorphous characteristics. Najmoddin et al. [N. Najmoddin, A.Beitollahi, M. Muhammed, N. Ansari, E. Devlin, S. Majid Mohseni, H.Rezaie, D. Niarchos, J. Akerman, M. S. Toprak, Effect of nanoconfinementon the formation, structural transition and magnetic behavior ofmesoporous copper ferrite, Journal of Alloys and Compounds 598 (2014)191-197, incorporated herein by reference in its entirety] has reportedthat cubic phase of CuFe₂O₄ can be effectively stabilized inside thepore channels of silica due to the suppression of John-Tellerdistortion. In addition, based on the characteristic support studies(XRD and TEM), a significant contribution of silica cages was confirmedin maintaining the super magnetic property at room temperature.

The surface area and pore size distributions of HYPS and CuFe₂O₄/HYPSwere analyzed using nitrogen adsorption technique. FIGS. 4A and 4B showthe isotherms (adsorption and desorption) and pore size distributionsfor (a) HYPS and (b) 30% CuFe₂O₄/HYPS, respectively. Monodispersedspherical silica HYPS shows a type IV isotherm corresponding to thepresence of mesopores. The silica hysteresis loop tends to be present athigher relative pressures of p/p₀>0.8. HYPS exhibits a surface area of170 m²/g, pore volume of 0.35 cm³/g with intermediate average pore sizedistributions of 8.3 nm. After spinel loading, about 28% reduction inBET surface area was observed, while a significant increase in the poresize from 8.3 nm to 16.0 nm was seen. The trend clearly shows theaccumulation of spinel ferrite nanoparticles at the external pores ofHYPS.

TABLE 1 Textural characteristics of HYPS and 30 wt % spinelferrites/HYPS. BJH Spinel BET adsorption Ferrite Surface cumulative PoreLoading area surface area volume Sample (wt %) (m²/g) (m²/g) (cm³/g) PD(nm) HYPS — 170 85 0.35 8.3 CuFe₂O₄/HYPS 30 47 45 0.18 16.0 NiFe₂O₄/HYPS30 29 30 0.12 17.1 MnFe₂O₄/HYPS 30 21 21 0.10 18.4

The surface morphology of parent HYPS and 30% CuFe₂O₄/HYPS was analyzedthrough SEM at two different scale bars of 3 and 1.0 μm (FIGS. 5A and5B). Parent HYPS showed the presence of monodispersed spherical silicasdistributed uniformly in the range of about 80 nm (FIG. 5A). Afterspinel loading, a different surface morphology was observed. The parentnanometer sized spherical spheres were found to be impregnated by nanospinels that were interrelated with each other (FIG. 5B). The CuFe₂O₄nanoparticles were microscopically captured using EDX-mapping analysisto observe the location of Cu and iron oxide over HYPS support. Themapping of HYPS showed homogeneous silica particles consistence with theSEM analysis. In case of copper ferrite nanoparticles, a uniformdistribution of Cu and Fe indicates mixed metal oxide formationshomogeneously spread over silica support (FIGS. 5E and 5F). In addition,additional small nanoclusters of Cu species were also observed.

The FT-IR spectra of silica HYPS showed several peaks corresponding toSi—O—Si stretching and vibration, hydroxyl, and Si—O bond at around 432cm⁻¹, 800 cm⁻¹, and between 900-1055 cm⁻¹, respectively. Notably, thepeak intensity of silica at around 432 cm⁻¹ and 900-1055 cm⁻¹corresponding to silanol groups decreased after CuFe₂O₄ deposition (FIG.6 ). Such pattern indicates the external surface occupation of spinelover HYPS. The peak position of 30% CuFe₂O₄/HYPS shows rearrangements ofpeak position at about 487 cm⁻¹ with tetrahedral and octahedral sites ofMFe₂O₄. The absorption band at 580 cm⁻¹ clearly shows the presence ofFe—O bond of spinel present over HYPS support.

The magnetic properties of spinel ferrite/HYPS nanocomposites (i.e.nanoformulations) were measured using vibrating sample magnetometer(VSM). FIG. 7 shows an overlay of VSM of a) 30% CuFe₂O₄/HYPS, (b) 30%NiFe₂O₄/HYPS, (c) 30% MnFe₂O₄/HYPS, and (d) 30% CoFe₂O₄/HYPS,respectively. Based on the composition of spinel ferrites, differentmagnitudes of magnetization were observed. The magnetization generatedby various spinel ferrites over HYPS was in the following order: 30 wt %CoFe₂O₄/HYPS (14.15 emug⁻¹)>30 wt % NiFe₂O₄/HYPS (7.73 emug⁻¹)>30 wt %CuFe₂O₄/HYPS (7.65 emug⁻¹)>30 wt % MnFe₂O₄/HYPS (1.49 emug⁻¹). Reducingthe particle size tends to decrease the magnetization saturation due tononcollinear spin arrangements at or near the surface of particles [B.Martinez, X. Obradors, L I. Balcells, A. Rouanet, C. Monty, Lowtemperature surface spin-glass transition in γ-Fe₂O₃ nanoparticles,Phys. Rev. Lett. 80 (1) (1998) 181-183, incorporated herein by referencein its entirety], which in turn influences the response of material tothe external magnetic field. The spinel properties are influenced by thecation distribution over the A and B sites, as presence of differentcations tends to influence the magnetic and electrical property. Copperferrites are known ferrites, where Zn atoms are substituted at thetetrahedral site of Cu, for variable magnetization property. In thepresent case, 30 wt % CuFe₂O₄/HYPS showed paramagnetic behavior, whilecobalt ferrite/silica showed ferromagnetism, which are reported to occurdue to anti parallel spins of Fe³⁺ located at tetrahedral sites, whileM²⁺ at octahedral sites. It has been shown that presence of small sizednanoclusters at the walls of hexagonal shaped MCM-41 tends to form superparamagnetic interactions among Fe³⁺ species, while large nanoclusterscontribute towards ferromagnetic property [S. Dasari, P. B. Tchounwou.Cisplatin in cancer therapy: molecular mechanisms of action. Eur JPharmacol. 740 (2014) 364-378, incorporated herein by reference in itsentirety]. In the present study, nickel ferrite/HYPS showed mostpronounced super paramagnetic behavior with narrow hysteresis followedby copper ferrite/HYPS. Such trend demonstrates the formation of smallnanosized nickel and copper spinel clusters over HYPS (FIG. 7 , lines“a” and “b”). MnFe₂O₄/HYPS showed the super paramagnetic behavior butwith much lower magnetization (FIG. 7 , line “c”). CoFe₂O₄/HYPSgenerated the highest magnetization with a broad hysteresis loopindicating a shift towards ferromagnetic behaviour compared to otherthree nanocomposites. Such magnetic trend shows the formation of largenanoclusters (FIG. 7 , line “d”).

Example 10

Results and Discussions: Nanotherapeutic

FIG. 8 summarizes the cisplatin adsorption capacity and percentagecumulative release in tumor at pH 5 for 24 h over 30% CuFe₂O₄/HYPS, 30%NiFe₂O₄/HYPS, 30% MnFe₂O₄/HYPS and 30% CoFe₂O₄/HYPS, respectively. Incase of cisplatin adsorption, 4 nanoformulations showed an adsorptionbetween 86-90%. In particular, CoFe₂O₄/HYPS and CuFe₂O₄/HYPS showedadsorption of about 86.5% and 88.6%, respectively. On the other hand,MnFe₂O₄/HYPS and NiFe₂O₄/HYPS showed a greater adsorption of 87.2% and90.3%, respectively. The cisplatin release profile calculated based onmmol of cisplatin per gram of nano support showed a significantvariation with respect to cisplatin release. For instance, thoughcisplatin adsorption remains similar among nanoformulations,NiFe₂O₄/HYPS and CuFe₂O₄/HYPS showed the highest cisplatin release,which was followed by MnFe₂O₄/HYPS and CoFe₂O₄/HYPS.

The drug release profile of spinel ferrite-based system MFe₂O₄═Ni, Co,Cu and Mn) was studied at simulated tumor acidic pH conditions (pH 5) at37° C. for 72 h (FIG. 9 ). The spinel ferrites studied include 4 typesof metal composites Ni, Co, Cu and Mn. The ratio of cisplatin (mmol) pergram of spinel HYPS nanosupport was maintained at 0.15. Among thedifferent nanoformulations, the order of cisplatin drug release rate wasin the following order: 30 wt % CuFe₂O₄/HYPS>30 wt % NiFe₂O₄/HYPS>30 wt% MnFe₂O₄/HYPS˜30 wt % CoFe₂O₄/HYPS, respectively. 30 wt % CuFe₂O₄/HYPSshowed the highest percentage cumulative cisplatin release of 90% within72 h. The study shows that fabrication of HYPS with 30 wt % CuFe₂O₄ isnot affecting cisplatin release at acidic tumor condition. 30 wt %NiFe₂O₄/HYPS showed the second-best formulation over HYPS. Thepercentage release was lower at about 61%, which indicates the positiveeffect of CuFe₂O₄/HYPS with respect to cisplatin release rate. Thistrend signifies the importance of synergism among CuFe₂O₄, cisplatin,and HYPS support, which facilitates releasing cisplatin efficiently(FIG. 9 ). However, cisplatin over Mn and Co based spinel ferrite showedan initial burst release of 75% within about 30 min, which then reducedto 53% at 72 h. This clearly indicates that in addition to CuFe₂O₄/HYPS,NiFe₂O₄/HYPS formulation can be another potential nanocarrier for drugdelivery application.

In order to understand the high cisplatin release ability of 30 wt %CuFe₂O₄/HYPS, the coordination environment of cisplatin over 30 wt %CuFe₂O₄/HYPS (active sample) and 30 wt % MnFe₂O₄/HYPS (in active sample)were analyzed using diffuse reflectance spectroscopy. Ferrites are cubicspinels containing tetrahedral and octahedral crystalline sites [N.Najmoddin, A. Beitollahi, E. Devlin, H. Kavas, S. M. Mohseni, J.Akerman, D. Niarchos, H. Rezaie, M. Muhammed, M. S. Toprak, Magneticproperties of crystalline mesoporous Zn-substituted copper ferritesynthesized under nanoconfinement in silica matrix, Microporous andMesoporous Materials 190 (2014) 346-355]. Both support samples beforecisplatin adsorption showed wide and strong absorption between 200-700nm, characteristics of spinel structure (FIG. 10 lines “a” and “c”).Remarkably, after platinum adsorption CuFe₂O₄/HYPS sample showedenhanced peak absorption up to 700 nm, while no significant improvementin the coordination site was observed over MnFe₂O₄/HYPS (FIG. 10 , lines“b” and “d”). In particular, a small absorption at around 224 nm showedthe presence of tetrahedral coordinated Pt nanoclusters, while asignificant enhancement of absorption peak at 350-600 nm shows thepresence of octahedral coordinated Pt species (FIG. 10 , line “b”). Thepresence of such strong bonds of tetrahedral and octahedral Pt speciesindicates high dispersity and interaction of Pt on HYPS silica support.Previously, it was shown that such Pt species over mesosilicaliteexerted high cytotoxic effect against HeLa (LC₅₀=0.02 mg/ml), MCF-7(LC₅₀=0.05 mg/ml), while less toxic towards normal fibroblast cells(LC₅₀=0.5 mg/ml) [Jermy B R, Acharya S, Ravinayagam V, Alghamdi H S,Akhtar S, Basuwaidan R S (2018) Hierarchical mesosilicalitenanoformulation integrated with cisplatin exhibits target-specificefficient anticancer activity. Appl Nanosci 8, 1205-1220, incorporatedherein by reference in its entirety]. Therefore, the activity ofnanotherapeutic (cisplatin loaded 30 wt % CuFe₂O₄/HYPS) against MCF-7cell line was studied using MTT assay.

Example 11

In Vitro Anti-Cancer Studies

To investigate cytotoxic efficiency of cisplatin-loaded/CuFe₂O₄-coatedsilica nanoparticles, cell viability using the MTT assay were assessed.Healthy cells are able to reduce MTT to the purple-colored formazan,while unhealthy/dead cells cannot. MCF7 cells were treated with thefollowing conditions: group A (CuFe₂O₄), group B (silica nanoparticles),group C (silica+CuFe₂O₄), group D (cisplatin), group E(cisplatin+silica+CuFe₂O₄), and group F(cisplatin+silica+CuFe₂O₄+chitosan) for 48 h (FIGS. 11 and 12A-G).CuFe₂O₄, silica, and their combination (groups A, B, and C) did not havea significant effect on cell viability. As expected, the pure cisplatingroup (D) had a significant reduction in cell viability, which reached53.8% at the lowest concentrations used. Cisplatin was able to maintaina steady reduction in cell viability as its concentration increased.Interestingly, when cisplatin was loaded into CuFe₂O₄-coated silicananoparticles (group E), a significant reduction in cell viability wasobserved as well. Similar to group D, group E showed a dose dependentreduction in cell viability that reached 56.5%, 48.1%, and 31.4% at0.05, 0.1, and 0.5 mg/mL, respectively. However, the chitosan-coatednanoparticles did not have any significant reduction in cell viability.The half maximal effective concentration (EC₅₀) was calculated from theline equation of each condition (FIG. 12G). These results show that thecisplatin-loaded/CuFe₂O₄-coated silica nanoparticles can effectivelyreduce the viability of human breast cancer cell line MCF7, thus makingit a promising option for drug delivery.

Cells were treated with the following conditions for 48 hours: group A(CuFe₂O₄), group B (silica nanoparticles), group C (silica+CuFe₂O₄),group D (cisplatin), group E (cisplatin+silica+CuFe₂O₄), and group F(cisplatin+silica+CuFe₂O₄+chitosan). For groups B, C, E, and F,treatment concentrations were as follows: 0.025, 0.05, 0.1, and 0.5mg/ml. To accurately reflect the concentration of CuFe₂O₄ (group A), andcisplatin (group D) that is encapsulated within these nanoparticles, thedrug loading experiments were used to calculate the actual concentrationof each in the other groups. Therefore, treatment concentrations used inthis experiment for group A were as follows: 0.0084, 0.0168, 0.0336, and0.168 mg/ml. Treatment concentrations used in this experiment for groupD were as follows: 0.001125, 0.00225, 0.0045, 0.0225 mg/ml. n=4independent experiments. Dashed line represents untreated control. Errorbars±S.E.M. * p<0.05; ** p<0.01; *** p<0.001; ****p<0.0001 versuscontrol using two-way ANOVA with Dunnett's post hoc testing.

Here, the cytotoxic efficiency of our nanoparticles was tested on thebreast cancer cell line MCF7. While CuFe₂O₄, silica, and theircombination were not cytotoxic, the cisplatin and thecisplatin-loaded/CuFe₂O₄-coated silica nanoparticles significantlyreduced cell viability. The cisplatin containing groups (D and E) showeda dose dependent response. The cisplatin-loaded/CuFe₂O₄-coated silicananoparticles (group E) caused a reduction in viability that reached56.5%, 48.1%, and 31.4% at 0.05, 0.1, and 0.5 mg/mL, respectively.

Phadatare et al. [M. R. Phadatare, V. M. Khot, A. B. Salunkhe, N. D.Thorat, S. H. Pawar, Studies on polyethylene glycol coating on NiFe₂O₄nanoparticles for biomedical applications, Journal of Magnetism andMagnetic Materials, 324 (2012) 770-772] has reported the preparation ofNiFe₂O₄ using combustion technique followed by polyethylene glycolcoating to improve the biocompatibility. The formation of agglomeratedfoam like morphology was observed for NiFe₂O₄ with a high saturationmagnetization value of 35 emug⁻¹. Sardrolhosseini et al. [A. R.Sardrolhosseini, M. Naseri, S. A. Rashid,Polypyrrole-chitosan/nickel-ferrite nanoparticle composite layer fordetecting heavy metal ions using surface plasmon resonance technique,Optics and Laser Technology 93 (2017) 216-223] reported the preparationof NiFe₂O₄ nanoparticle composite involving polypyrrole chitosan throughelectrochemical polymerization technique. In the present disclosure,efforts were made toward increasing the biocompatibility of 30 wt %CuFe2O4/HYPS by wrapping with chitosan. However, EC₅₀ value of F showsthat wrapping cisplatin/30 wt % CuFe2O4/HYPS composite with chitosan(Group F) showed no significant effect on cell viability compared withGroup D and E. Initially, the cisplatin (mmol) per gram of CuFe₂O₄/HYPSnanosupport was maintained at 0.15. During chitosan wrapping process,the initial pH of the chitosan solution was maintained at 2.77, whichthen increased to 6.4 by dropwise addition of 1 M NaOH solution. AfterpH adjustment, 1 g cisplatin loaded CuFe₂O₄/HYPS was added and themixture was kept under stirring for 24 hours, then the pH was increasedto 7. The mixture was kept under stirring for another 24 hours, thencentrifuged, washed and dried under vacuum for 48 h at 37° C. Theanalysis of filtered solution showed a decrease in the mmolcisplatin/gram of CuFe₂O₄/HYPS from 0.15 to 0.03. This shows thatcisplatin during chitosan pH adjustment step from 6.4 to 7 prematurelyrelease cisplatin to the solution and therefore Group F showed lessinhibitory effect compared to Group D and Group E. However, the presentdisclosure shows that Group E itself is the best nanoformulation thatcan be further tuned for chitosan loading by adjusting the cisplatinloading step after chitosan wrapping. In this way, the cisplatin releaseduring chitosan pH adsorption process can be negated, whilebiocompatible of the nanocomposite will be increased. These results showthat cisplatin-loaded/CuFe₂O₄-coated silica nanoparticles caneffectively target cancerous cells. It also shows that CuFe₂O₄-coatedsilica nanoparticles could be a useful drug delivery system.

Overall, spinel ferrite/HYPS hybrid nanoformulations (FIG. 13 ) wereexplored to load the multifunctional magnetic silica drug deliveryvehicle with deliverable cisplatin drug. 30% of different spinel MFe₂O₄(M=Cu, Ni, Co, and Mn) has been loaded over HYPS through dryimpregnation technique. The presence of cubic spinel containingtetrahedral and octahedral crystalline sites was confirmed through XRD(FIG. 1 ), FT-IR (FIG. 6 ), and DR UV-vis analysis (FIG. 10 ). Theisotherm patterns of HYPS show that after spinel impregnation, anon-significant surface occupation (about 28%) occurs, leaving space foreffective functionalization of cisplatin and chitosan wrapping. Thecumulative pore volume showed about 50% occupation compared to HYPS(FIG. 4B). EDX-mapping analysis showed the presence of homogeneoussilica particles, with uniform distribution of Cu and Fe mixed oxidephase of copper ferrite over HYPS silica (FIGS. 5E, 5F). VSM analysis of30 wt % CuFe₂O₄/HYPS showed paramagnetic behavior, while cobaltferrite/silica showed ferromagnetism (FIG. 7 ). The drug release profileof various spinel ferrite-based systems showed 30 wt % CuFe₂O₄/HYPShaving the highest percentage cumulative cisplatin release of 90% for 72h (FIGS. 8 and 9 ). Such high cisplatin release may be due to thepresence of tetrahedral coordinated Pt nanoclusters, and octahedralcoordinated Pt species (FIG. 10 ). IC₅₀ value of experimental design inin vitro study and percentage cell viability using MTT assay on MCF-7cell line showed significant inhibitory effect of developed cisplatin/30wt % CuFe₂O₄/HYPS nanocomposite, while development of chitosan wrappingstep might be vital to further increase the biocompatibility (FIGS. 11and 12A-G).

Example 12

An effective multifunctional spinel ferrite/silica nanocomposite systemhas been developed through dry impregnation technique. Among differentspinel materials MFe₂O₄ (M=Cu, Ni, Co, and Mn), CuFe₂O₄/HYPSnanocomposite showed high cisplatin delivery and anticancer efficacy dueto synergetic interactions between Pt and CuFe₂O₄. The presence of cubiccopper ferrite was confirmed with uniform distribution of Cu and Femixed oxide phase over HYPS. The order of cisplatin drug release was inthe following order: 30 wt % CuFe₂O₄/HYPS>30 wt % NiFe₂O₄/HYPS>30 wt %MnFe₂O₄/HYPS 30 wt % CoFe₂O₄/HYPS

The low-cost spinel ferrites in presence of spherical silica have shownsuperior magnetic behavior, reduced aggregation and toxicity. Thepresence of proper surface area of HYPS helps generating magneticallyactive species and facilitates efficient cisplatin loading-releasecapabilities with targeted anticancer efficiency against cancer cellline MCF-7 in vitro.

Treatment of MCF-7 cancerous cell line with cisplatin/30 wt %CuFe₂O₄/HYPS (Group-E) showed high cell killing activity compared to 30wt % CuFe₂O₄/HYPS-chitosan (Group-F). Overall, superparamagnetic copperferrite/HYPS is a potential candidate for multifunctional theranosticapplications for treating deadly diseases. The nanoformulation withmagnetic property of spinel loaded over monodisperse silica can befurther manipulated with drugs, antioxidants, and biocompatiblepolymers.

The invention claimed is:
 1. A method for treating a proliferativedisorder, the method comprising administering a nanotherapeutic to asubject in need of therapy, wherein the nanotherapeutic comprises: ananoformulation comprising: mesoporous silica nanoparticles in the formof monodispersed spherical silica having a particle size distribution ofabout 80 nm; and spinel ferrite nanoclusters of formula (I)MFe₂O₄  (I) wherein M is at least one transition metal element selectedfrom the group consisting of Cu and Ni; wherein: the spinel ferritenanoclusters are impregnated on the mesoporous silica nanoparticles; andthe spinel ferrite nanoclusters are present in an amount of 25-35 wt %relative to a total weight of the nanoformulation, and cisplatinencapsulated within pores of the nanoformulation, wherein thenanoformulation has a cisplatin release at a pH 5 at 37° C. for 72 h of60-80 wt. %; wherein the pores of the nanoformulation have a porediameter in a range of 10-25 nm, and the nanoformulation has a porevolume in a range of 0.05 0.3 cm³/g, a BET surface area in a range of15-70 m²/g, and a saturation magnetization value in a range of 7-15emu/g; and wherein the proliferative disorder is a cancer selected fromthe group consisting of breast cancer, ovarian cancer, cervical cancer,testicular cancer, colon cancer, bladder cancer, and lung cancer.
 2. Themethod of claim 1, wherein the nanotherapeutic further comprises one ormore pharmaceutically acceptable carriers and/or excipients selectedfrom the group consisting of a buffer, an inorganic salt, a fatty acid,a vegetable oil, a synthetic fatty ester, a surfactant, and a polymer.3. The method of claim 1, wherein the cisplatin is present at aconcentration of 0.01-10 mmol/g relative to a total weight of thenanoformulation.
 4. The method of claim 1, wherein 0.5-800 mg/kg of thenanotherapeutic is administered per body weight of the subject.
 5. Themethod of claim 1, wherein the cancer proliferative disorder is breastcancer.
 6. The method of claim 1, wherein the subject is a mammal.